Adeno-associated virus variant capsids and methods of use thereof

ABSTRACT

Provided herein are variant adeno-associated virus (AAV) capsid proteins having one or more modifications in amino acid sequence relative to a parental AAV capsid protein, which, when present in an AAV virion, confer increased infectivity of one or more types of muscle cells as compared to the infectivity of the muscle cells by an AAV virion comprising the unmodified parental AAV capsid protein. Also provided are recombinant AAV virions and pharmaceutical compositions thereof comprising a variant AAV capsid protein as described herein, methods of making these rAAV capsid proteins and virions, and methods for using these rAAV capsid proteins and virions in research and in clinical practice, for example in, e.g., the delivery of nucleic acid sequences to one or more muscle cells for the treatment of muscle disorders and diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the 35 U.S.C. 371 National Stage of InternationalApplication Number PCT/US2018/051812, filed Sep. 19, 2018, which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 62/560,901,filed Sep. 20, 2017, the full disclosures of which are incorporatedherein by reference.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled“090400-5009-WO-Sequence-Listing.txt”, created on or about Mar. 17,2020, with a file size of about 290,000 bytes contains the sequencelisting for this application and is hereby incorporated by reference inits entirety.

FIELD OF THE INVENTION

The invention disclosed herein relates generally to the field ofadeno-associated virus (AAV) virions comprising variant capsid proteinsand the generation of such variant capsids using directed evolutiontechniques.

BACKGROUND OF THE DISCLOSURE

Muscle is associated with a variety of severe genetic disorders. Muscleis the target tissue in gene therapy for many muscular dystrophydiseases and also can be exploited as a biofactory to produce secretoryfactors to treat systemic disease. Delivering therapeutic genes tomuscle tissue in human is arguably the most urgent unmet need intreating muscle-related diseases.

One approach to accomplish muscle-directed gene delivery is gene-basedadeno-associated virus (AAV)-mediated therapy, in which a recombinantadeno associated virus (rAAV) is used to deliver a gene to one or moremuscle cells, for example to replace a missing gene, to correct adominant defective gene, or to provide a template for continuous proteintherapy. While AAV-based clinical gene therapy has been increasinglysuccessful, it is still fraught with shortcomings with regard to viralvector properties, including, for example, targeting the desired cellsof the muscle with high efficiency. Accordingly, there is a need in theart for new AAV variants with superior transduction capabilities thatwill provide for more effective gene-based delivery to the cells of themuscle for the treatment of disease. There is a need in the art for suchAAV variants which exhibit an enhanced muscle transduction profile—insome instances broadly, in other instances preferentially to certainmuscle cell types—as compared to wild-type AAVs and AAV variants asknown in the art.

Naturally occurring AAV is a single stranded DNA virus that containsthree open reading frames, rep, cap, and aap. The first gene, rep,encodes four proteins necessary for genome replication (Rep78, Rep68,Rep52, and Rep40), the second, cap, expresses three structural proteins(VP1-3) that assemble to form the viral capsid, and the third expressesthe assembly activating protein (AAP) that is essential for capsidassembly. AAV is dependent upon the presence of a helper virus, such asan adenovirus or herpesvirus, for active replication. In the absence ofa helper virus, AAV establishes a latent state in which its genome ismaintained episomally or integrated into the host chromosome in theAAVS1 locus.

In vitro and in vivo directed evolution techniques may be used to selectfor AAV variants that offer an improvement over current AAV-based genedelivery vectors. Such directed evolution techniques are known in theart and described, e.g., in PCT publication WO 2014/194132 and Kotterman& Schaffer (Nature Review Genetics, AOP, published online 20 May 2014;doi: 10.1038/nrg3742), both of which are incorporated herein in theirentirety by reference. Directed evolution is a capsid engineeringapproach that emulates natural evolution through iterative rounds ofgenetic diversification and selection processes, thereby enabling theaccumulation of beneficial mutations that progressively improve thefunction of a biomolecule such as an AAV-based virion. In this approach,wild-type AAV cap genes are diversified to create large geneticlibraries that are packaged to generate libraries of viral particles,and selective pressure is applied to isolate unique variants withsuperior phenotypes that can overcome gene delivery barriers.

AAV variants have been disclosed in, for example, U.S. Pat. Nos.9,193,956; 9,186,419; 8,632,764; 8,663,624; 8,927,514; 8,628,966;8,263,396; 8,734,809; 8,889,641; 8,632,764; 8,691,948; 8,299,295;8,802,440; 8,445,267; 8,906,307; 8,574,583; 8,067,015; 7,588,772;7,867,484; 8,163,543; 8,283,151; 8,999,678; 7,892,809; 7,906,111;7,259,151; 7,629,322; 7,220,577; 8,802,080; 7,198,951; 8,318,480;8,962,332; 7,790,449; 7,282,199; 8,906,675; 8,524,446; 7,712,893;6,491,907; 8,637,255; 7,186,522; 7,105,345; 6,759,237; 6,984,517;6,962,815; 7,749,492; 7,259,151; and 6,156,303; United StatesPublication Numbers 2013/0295614; 2015/0065562; 2014/0364338;2013/0323226; 2014/0359799; 2013/0059732; 2014/0037585; 2014/0056854;2013/0296409; 2014/0335054 2013/0195801; 2012/0070899; 2011/0275529;2011/0171262; 2009/0215879; 2010/0297177; 2010/0203083; 2009/0317417;2009/0202490; 2012/0220492; 2006/0292117; and 2004/0002159; EuropeanPublication Numbers 2692731 A1; 2383346 B1; 2359865 B1; 2359866 B1;2359867 B1; and 2357010 B1; 1791858 B1; 1668143 B1; 1660678 B1; 1664314B1; 1496944 B1; 1456383 B1; 2341068 B1; 2338900 B1; 1456419 B1; 1310571B1; 1456383 B1; 1633772 B1; and 1135468 B1; and International (PCT)Publication Numbers WO 2014/124282; WO 2013/170078; WO 2014/160092; WO2014/103957; WO 2014/052789; WO 2013/174760; WO 2013/123503; WO2011/038187; and WO 2008/124015; WO 2003/054197; however, none of thesereferences disclose the embodiments and/or features and/or compositionof matter structures of the AAV variants disclosed herein.

All documents and references cited herein and in the referenced patentdocuments, are hereby incorporated herein by reference.

SUMMARY OF THE INVENTION

Provided herein are variant adeno-associated virus (AAV) capsid proteinshaving one or more modifications in amino acid sequence relative to aparental AAV capsid protein, which, when present in an AAV virion,confer increased infectivity of one or more types of muscle cells ascompared to the infectivity of the muscle cells by an AAV virioncomprising an unmodified parental AAV capsid protein. Also provided arerecombinant AAV virions and pharmaceutical compositions thereofcomprising a variant AAV capsid protein as described herein, methods ofmaking variant rAAV capsid proteins and virions, and methods for usingthese rAAV capsid proteins and virions in research and in clinicalpractice, for example in the delivery of nucleic acid sequences to oneor more muscle cells for the treatment of disorders and diseases.

In some aspects of the disclosure, variant adeno-associated virus (AAV)capsid proteins are provided, these variant AAV capsid proteins havingone or more modifications in amino acid sequence relative to a parentalAAV capsid, which, when present in an AAV virion, confer increasedinfectivity of one or more types of muscle cells (e.g. skeletal musclecells and/or cardiac muscle cells) as compared to the infectivity of themuscle cells by an AAV virion comprising a parental AAV capsid proteinthat does not comprise the amino acid sequence modification. In relatedaspects of the disclosure, the variant AAV capsid proteins, when presentin an AAV virion also confer enhanced resistance to neutralization byanti-AAV antibodies.

In some aspects of the disclosure, recombinant AAV (rAAV) virions areprovided, these rAAV virions comprising a variant capsid protein asdescribed herein, wherein the rAAV virions exhibit increased infectivityof one or more types of muscle cells (e.g. skeletal muscle cells and/orcardiac muscle cells) relative to the infectivity of the muscle cell byan AAV virion comprising a corresponding unmodified parental AAV capsidprotein. In some embodiments, the rAAV virion exhibits increasedinfectivity of all muscle cells relative to the AAV virion comprisingthe parental AAV capsid protein. In other embodiments, the rAAV virionexhibits increased infectivity of certain muscle cell types but notothers relative of the AAV virion comprising the parental AAV capsidprotein. Put another way, the rAAV virion exhibits increased infectivitythat is preferential for certain muscle cell types but not others, e.g.the rAAV demonstrates a preferentially increased infectivity of one ormore cell types selected from skeletal muscle fibroblasts, skeletalmuscle satellite cells, cardiac fibroblasts, cardiac progenitor cells,smooth muscle cells and/or diaphragm muscle cells, but does notdemonstrate increased infectivity of all cell types.

In some embodiments, the rAAV virion comprises a heterologous nucleicacid. In some such embodiments, the heterologous nucleic acid encodes anRNA that encodes a polypeptide. In other such embodiments, theheterologous nucleic acid sequence encodes an RNA that does not encode apolypeptide, e.g. the heterologous nucleic acid sequence is an RNAinterference agent, a guide RNA for a nuclease, etc.

Also provided herein are pharmaceutical compositions comprising thesubject infectious rAAV virions and a pharmaceutically acceptablecarrier.

Also provided is the use of an rAAV virion comprising a variant capsidprotein as herein described in a method of delivering a heterologousnucleic acid to a target cell (such as a cardiomyocyte) by contactingthe target cell with the rAAV virion. In some embodiments, the targetcell is in vivo, such as in the heart of an individual in need oftreatment for a cardiovascular disorder. In other embodiments, thetarget cell is in vitro.

Also provided are methods of treating and/or preventing a disease (e.g.a cardiac or skeletal muscle disorder) by administering to a subject inneed of such treatment an effective amount of rAAV virions comprising avariant capsid protein as herein described or a pharmaceuticalcomposition comprising an effective amount of the rAAV virions.

Also provided is an isolated nucleic acid comprising a sequence encodinga variant AAV capsid protein as described herein and a host cellcomprising the isolated nucleic acid. In yet other embodiments, theisolated nucleic acid and/or isolated host cell comprises the rAAV.

In some aspects, the variant AAV capsid protein comprises an insertionof from about 5 amino acids to about 20 amino acids (a “heterologouspeptide”, or “peptide insertion”) in the GH-loop of the capsid protein,relative to a corresponding parental AAV capsid protein, wherein thevariant capsid protein, when present in an AAV virion, confers increasedinfectivity of a muscle cell compared to the infectivity of a musclecell by an AAV virion comprising the corresponding parental AAV capsidprotein. In some embodiments, the peptide comprises or consistsessentially of a sequence selected from the group consisting of NKIQRTD(SEQ ID NO:13), NKTTNKD (SEQ ID NO:14), TNKIGVT (SEQ ID NO:15), GNLTKGN(SEQ ID NO:16), NTVKLST (SEQ ID NO:17), SNTVKAI (SEQ ID NO:18), ASNITKA(SEQ ID NO:19), DNTVTRS (SEQ ID NO:20), NKISAKD (SEQ ID NO:21), NQDYTKT(SEQ ID NO:22), QADTTKN (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), SNTTQKT(SEQ ID NO:25), ASDSTKA (SEQ ID NO:26), LANKIQRTDA (SEQ ID NO:27),LANKTTNKDA (SEQ ID NO:28), LATNKIGVTA (SEQ ID NO:29), LAGNLTKGNA (SEQ IDNO:30), LANTVKLSTA (SEQ ID NO:31), LASNTVKAIA (SEQ ID NO:32), LAASNITKAA(SEQ ID NO:33), LADNTVTRSA (SEQ ID NO:34), LANKISAKDA (SEQ ID NO:35),LANQDYTKTA (SEQ ID NO:36), LATNKIGVTS (SEQ ID NO:37), LATNKIGVTA (SEQ IDNO:38), LAQADTTKNA (SEQ ID NO:39), LATNRTSPDA (SEQ ID NO:40), LASNTTQKTA(SEQ ID NO:41), and LAASDSTKAA (SEQ ID NO:42). In some preferredembodiments, the peptide comprises or consists essentially of a sequenceselected from the group consisting of NKIQRTD (SEQ ID NO:13), NKTTNKD(SEQ ID NO:14), TNKIGVT (SEQ ID NO:15), LANKIQRTDA (SEQ ID NO:27),LANKTTNKDA (SEQ ID NO:28), LATNKIGVTA (SEQ ID NO:29) and LATNKIGVTS (SEQID NO:37).

In some aspects, the variant AAV capsid protein comprises one or moreamino acid substitutions relative to a corresponding parental AAV capsidprotein, wherein the variant capsid protein, when present in an AAVvirion, confers increased infectivity of a muscle cell compared to theinfectivity of a muscle cell by an AAV virion comprising thecorresponding parental AAV capsid protein.

In some embodiments, a variant AAV capsid protein is disclosedcomprising a P363L substitution relative to AAV2 and optionally furthercomprising an E347K and/or V708I substitution relative to AAV2.

In related aspects, the variant AAV capsid protein comprises a peptideinsertion and one or more amino acid substitutions relative to acorresponding parental AAV capsid protein, wherein the variant capsidprotein, when present in an AAV virion, confers increased infectivity ofa muscle cell compared to the infectivity of a muscle cell by an AAVvirion comprising the corresponding parental AAV capsid protein. Inseveral embodiments, a variant AAV capsid protein is provided comprisinga peptide insertion and a V708I substitution relative to AAV2, whereinthe peptide insertion is optionally selected from the group consistingof NKIQRTD (SEQ ID NO:13), NKTTNKD (SEQ ID NO:14), TNKIGVT (SEQ IDNO:15), GNLTKGN (SEQ ID NO:16), NTVKLST (SEQ ID NO:17), SNTVKAI (SEQ IDNO:18), ASNITKA (SEQ ID NO:19), DNTVTRS (SEQ ID NO:20), NKISAKD (SEQ IDNO:21), NQDYTKT (SEQ ID NO:22), QADTTKN (SEQ ID NO:23), TNRTSPD (SEQ IDNO:24), SNTTQKT (SEQ ID NO:25), ASDSTKA (SEQ ID NO:26), LANKIQRTDA (SEQID NO:27), LANKTTNKDA (SEQ ID NO:28), LATNKIGVTA (SEQ ID NO:29),LAGNLTKGNA (SEQ ID NO:30), LANTVKLSTA (SEQ ID NO:31), LASNTVKAIA (SEQ IDNO:32), LAASNITKAA (SEQ ID NO:33), LADNTVTRSA (SEQ ID NO:34), LANKISAKDA(SEQ ID NO:35), LANQDYTKTA (SEQ ID NO:36), LATNKIGVTS (SEQ ID NO:37),LATNKIGVTA (SEQ ID NO:38), LAQADTTKNA (SEQ ID NO:39), LATNRTSPDA (SEQ IDNO:40), LASNTTQKTA (SEQ ID NO:41), and LAASDSTKAA (SEQ ID NO:42),preferably from the group consisting of NKIQRTD (SEQ ID NO:13), NKTTNKD(SEQ ID NO:14), TNKIGVT (SEQ ID NO:15), LANKIQRTDA (SEQ ID NO:27),LANKTTNKDA (SEQ ID NO:28), LATNKIGVTA (SEQ ID NO:29) and LATNKIGVTS (SEQID NO:37). In several embodiments, a variant AAV capsid protein isprovided comprising a peptide insertion and a P363L substitutionrelative to AAV2, wherein the peptide insertion is optionally selectedfrom the group consisting of GNLTKGN (SEQ ID NO:16), LAGNLTKGNA (SEQ IDNO:30), QADTTKN (SEQ ID NO:23) and LAQADTTKNA (SEQ ID NO:39).

In some embodiments, a variant AAV capsid protein is disclosedcomprising the heterologous peptide LANKIQRTDA (SEQ ID NO:27) and aV708I substitution relative to AAV2 and optionally further comprising anA593E and/or S109T and/or T330A and/or R588M substitution relative toAAV2. In other embodiments, a variant AAV capsid protein is disclosedcomprising the heterologous peptide LANKIQRTDA (SEQ ID NO:27) and anA35P substitution relative to AAV2. In other embodiments, a variant AAVcapsid protein is disclosed comprising the heterologous peptideLANKIQRTDA (SEQ ID NO:27) and amino acid substitutions N312K, N449D,N551S, I698V, and L735Q relative to AAV2 and optionally furthercomprising a V708I substitution relative to AAV2.

In some embodiments, a variant AAV capsid protein is disclosedcomprising the heterologous peptide LANKTTNKDA (SEQ ID NO:28) and aV708I substitution relative to AAV2 and optionally further comprising anS109T and/or W694C and/or W606C substitution relative to AAV2. In otherembodiments, a variant AAV capsid protein is disclosed comprising theheterologous peptide LANKTTNKDA (SEQ ID NO:28) and an I698V substitutionrelative to AAV2. In other embodiments, a variant AAV capsid protein isdisclosed comprising the heterologous peptide LANKTTNKDA (SEQ ID NO:28)and amino acid substitutions N312K, N449D, N551S, I698V, and L735Qrelative to AAV2 and optionally further comprising a V708I substitutionrelative to AAV2.

In some embodiments, a variant AAV capsid protein is disclosedcomprising the heterologous peptide LATNKIGVTA (SEQ ID NO:29) and aV708I substitution relative to AAV2 and optionally further comprising anN449K and/or G222S substitution relative to AAV2. In other embodiments,a variant AAV capsid protein is disclosed comprising the heterologouspeptide LATNKIGVTA (SEQ ID NO:29) and amino acid substitutions N312K,N449D, N551S, I698V, and L735Q relative to AAV2 and optionally furthercomprising a V708I substitution relative to AAV2.

In some embodiments, a variant AAV capsid protein is disclosedcomprising a heterologous peptide as described herein and a P363Lsubstitution relative to AAV2.

Also disclosed herein are methods for manufacture and/or delivery of anrAAV comprising a variant AAV capsid as disclosed herein. In addition,provided herein are kits comprising an rAAV comprising a variant AAVcapsid as disclosed herein and for use in methods described herein.

In other embodiments, the AAV virion comprising the variant capsidprotein in the preceding paragraphs may incorporate any of the precedingor subsequently disclosed embodiments. Indeed, it is appreciated thatcertain features of the invention, which are, for clarity, described inthe context of separate embodiments, may also be provided in combinationin a single embodiment. Conversely, various features of the invention,which are, for brevity, described in the context of a single embodiment,may also be provided separately or in any suitable sub-combination. Allcombinations of the embodiments pertaining to the invention arespecifically embraced by the invention and are disclosed herein just asif each and every combination was individually and explicitly disclosed.In addition, all sub-combinations of the various embodiments andelements thereof are also specifically embraced by the invention and aredisclosed herein just as if each and every such sub-combination wasindividually and explicitly disclosed herein.

The Summary of the Invention is not intended to define the claims nor isit intended to limit the scope of the invention in any manner.

Other features and advantages of the invention disclosed herein will beapparent from the following Figures, Detailed Description, and theClaims.

BRIEF DESCRIPTION OF THE FIGURES

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1 depicts embodiments of a directed evolution methodology. Step (a)depicts the generation of a viral capsid library comprising combinationsof DNA mutation techniques and cap genes. Step (b) depicts the packagingof the viruses such that each viral particle is composed of a mutantcapsid surrounding the cap gene encoding that capsid and purified. Thecapsid library is then placed under selective pressure in vitro or invivo. In this aspect of the directed evolution technology, tissues orcellular material of interest are harvested for isolation of AAVvariants that have successfully infected that target, and the successfulviruses are recovered. Step (c) depicts the Stage 1 enrichment ofsuccessful clones through repeated selection. Step (d) depicts the Stage2 enrichment of selected cap genes which undergo re-diversification andfurther selection steps to iteratively increase viral fitness. Step (e)depicts the variants, identified as hits during Vector Selection Stages1 and 2, which will be manufactured as recombinant AAV vectors andcharacterized for the level of transduction of various cell types andtissue targets. By the nature of the AAV directed evolution process,variants that are disclosed herein have already demonstrated the abilityto transduce muscle cells and deliver a genome (the genome encoding thevariant cap gene) during the selection process.

FIG. 2 shows PCR amplification of viral genomes from the heart andskeletal muscle tissues from a representative round of selection. Bandswithin red boxes represent successful amplification of viral genomes.

FIGS. 3A-3C show frequency of motifs within sequencing analysis. FIG. 3Aprovides Round 4 sequencing analysis for the selective pressure ofintravenous delivery to cardiac tissue. FIG. 3B provides Round 2sequencing analysis for the selective pressure of intravenous deliveryin the presence of neutralizing antibodies to cardiac tissue. FIG. 3Cprovides Round 3 sequencing analysis for the selective pressure ofintravenous delivery to skeletal muscle tissue. FIG. 3A shows 57.40%LANKIQRTDA (SEQ ID NO: 27) Motif, 16.96% LANKTTNKDA (SEQ ID NO: 28)Motif, 7.32% A593E Motif, 7.32% Other, 4.88% V708I Motif and 4.88%LASNTVKAIA (SEQ ID NO: 32) Motif. FIG. 3B shows 21.14% Other, 20.33%LAQADTTKNA (SEQ ID NO: 39) Motif, 15.45% LANKTTNKDA (SEQ ID NO: 28)Motif, 15.45% LAASNITKAA (SEQ ID NO: 33) Motif, 15.45% AAV6/AAV5 ChimeraMotif and 12.20% LANTVKLSTA (SEQ ID NO: 31) Motif. FIG. 3C shows 43.21%A593E Motif, 41.98% P363L Motif and 14.81% Other.

FIGS. 4A-4C FIG. 4A is a representative three-dimensional model of AAV2containing a random heptamer following amino acid 587 and a V708Isubstitution. FIG. 4B is a representative three-dimensional model of theAAV6/AAV5 chimera containing V229I, A490T, and A581T substitutions(corresponding to the amino acid sequence set forth as SEQ ID NO:62).FIG. 4C is a representative three-dimensional model of AAV2 containing aP363L substitution.

FIG. 5 provides an alignment of wild-type AAV SEQ ID NOS:1-11 showingamino acid locations between and across the wild-type (naturallyoccurring) serotypes AAV1, AAV2, AAV3A, AAV3B, and AAV4-10.

FIGS. 6A-6E provide data on the transduction of human cardiomyocytes invitro by recombinant AAV virus comprising the novel AAV variantLANKIQRTDA+V708I (SEQ ID NO:43) capsid, the novel AAV variantLANKTTNKDA+V708I (SEQ ID NO:48) capsid, and the novel LATNKIGVTA+V708I(SEQ ID NO:46) variant capsid, each expressing a GFP transgene under thecontrol of the CAG promoter. FIG. 6A: Cells that were differentiatedinto cardiomyocytes from a human pluripotent stem cell line wereinfected with novel AAV variant LANKIQRTDA+V708I (SEQ ID NO:43).CAG.GFP,novel AAV variant LANKTTNKDA+V708I (SEQ ID NO:48).CAG.GFP, novel AAVvariant LATNKIGVTA+V708I (SEQ ID NO:46).CAG.GFP or wild type controlsAAV1.CAG.GFP, AAV2.CAG.GFP, and AAV9.CAG.GFP at MOIs of 20, 100, 500,and 2500. Immunofluorescence imaging of the cell cultures 6 days afterinfection at all MOIs demonstrate that the novel AAV variant capsidstransduce cardiomyocytes better than wild type AAV1, AAV2, or AAV9capsids. FIG. 6B: Quantification of the percent of GFP-positivecardiomyocytes in each culture by flow cytometry reveals that the novelAAV variant capsids provide for a significant, dose-dependentimprovement in the number of cells transduced over wild type AAV1, AAV2,or AAV9 capsids. *p<0.05 FIGS. 6C-6D: Quantification of the amount ofGFP in each culture by Western blot reveals that the novel AAV variantsprovide for significant improvement in expression of the transgene overwild type AAV1, AAV2, or AAV9 capsids. NT=not transduced. FIG. 6E: Cellsthat were differentiated into cardiomyocytes from a human pluripotentstem cell line were infected with novel AAV variant LANKIQRTDA+V708I(SEQ ID NO:43).CAG.GFP, novel AAV variant LANKTTNKDA+V708I (SEQ IDNO:48).CAG.GFP, novel AAV variant LATNKIGVTA+V708I (SEQ IDNO:46).CAG.GFP or wild type controls AAV1.CAG.GFP, AAV2.CAG.GFP, andAAV9.CAG.GFP. Immunofluorescence imaging of the cell cultures on days 1,2, 3, and 5 after infection at an MOI of 500 demonstrate that the novelAAV variant capsids transduce cardiomyocytes better and begin expressingthe GFP transgene earlier than wild type AAV1, AAV2, or AAV9 capsids.

FIGS. 7A-E provide data on the transduction of human cardiomyocytes invitro by recombinant AAV virus comprising the novel AAV variantAAV6/AAV5 chimera capsid of SEQ ID NO: 62, expressing a GFP transgeneunder the control of the CAG promoter. FIG. 7A: Cells that weredifferentiated into cardiomyocytes from a human pluripotent stem cellline were infected with novel AAV variant AAV6/AAV5 chimera capsid orwild type controls AAV1.CAG.GFP, AAV8.CAG.GFP, and AAV9.CAG.GFP at MOIsof 100, 500, and 2500. Immunofluorescence imaging of the cell cultures 6days after infection at all MOIs demonstrate that the novel AAV variantcapsid transduces cardiomyocytes better than wild type AAV1, AAV8, orAAV9 capsids. FIG. 7B: Quantification of the percent of GFP-positivecardiomyocytes in each culture by flow cytometry reveals that the novelAAV variant capsid provides for a significant, dose-dependentimprovement in the number of cells transduced over wild type AAV1, AAV8,or AAV9 capsids. *p<0.05 FIGS. 7C-7D: Quantification of the amount ofGFP in each culture by Western blot reveals that the novel AAV variantprovides for significant improvement in expression of the transgene overwild type AAV1, AAV8, or AAV9 capsids. vehicle=not transduced. FIG. 7E:Cells that were differentiated into cardiomyocytes from a humanpluripotent stem cell line were infected with novel AAV variantAAV6/AAV5 chimera capsid or wild type control AAV8.CAG.GFP.Immunofluorescence imaging of the cell cultures on days 3, 4, 5, and 6after infection at an MOI of 2500 demonstrate that the novel AAV variantcapsids transduce cardiomyocytes better and begin expressing the GFPtransgene earlier than the wild type AAV8 capsid.

FIGS. 8A-C provide data on the transduction of human skeletal myofibersin vitro by recombinant AAV virus comprising the novel AAV variantLANKIQRTDA+V708I (SEQ ID NO:43) capsid, the novel AAV variantLANKTTNKDA+V708I (SEQ ID NO:48) capsid, and the novel AAV variantAAV6/AAV5 chimera capsid, each expressing a GFP transgene under thecontrol of the CAG promoter. FIG. 8A: Cells that were differentiatedinto skeletal myofibers from human primary myoblasts were infected withnovel AAV variant LANKIQRTDA+V708I (SEQ ID NO:43).CAG.GFP, novel AAVvariant LANKTTNKDA+V708I (SEQ ID NO:48).CAG.GFP, novel AAV variantAAV6/AAV5 chimera.CAG.GFP or wild type controls AAV8.CAG.GFP andAAV9.CAG.GFP at MOIs of 100, 500, and 2500. Immunofluorescence imagingof the cell cultures 7 days after infection at all MOIs demonstrate thatthe novel AAV variant capsids transduce skeletal myofibers better thanwild type AAV8 or AAV9 capsids. FIG. 8B: Quantification of the percentof GFP-positive skeletal myofibers in each culture by flow cytometryreveals that the novel AAV variant capsids provide for a significant,dose-dependent improvement in the number of cells transduced over wildtype AAV8 or AAV9 capsids. *p<0.05 FIG. 8C: Cells that weredifferentiated into skeletal myofibers from human primary myoblasts wereinfected with novel AAV variant LANKIQRTDA+V708I (SEQ ID NO:43).CAG.GFP,novel AAV variant AAV6/AAV5 chimera.CAG.GFP or wild type controlsAAV8.CAG.GFP and AAV9.CAG.GFP. Immunofluorescence imaging of the cellcultures on days 2-7 after infection at an MOI of 2500 demonstrate thatthe novel AAV variant capsids transduce skeletal myofibers better andbegin expressing the GFP transgene earlier than wild type AAV8 or AAV9capsids.

FIGS. 9A-B provide data on the transduction of human muscle progenitorcells in vitro by recombinant AAV virus comprising the novel AAV variantLANKIQRTDA+V708I (SEQ ID NO:43) capsid, the novel AAV variantLANKTTNKDA+V708I (SEQ ID NO:48) capsid, and the novel AAV variantAAV6/AAV5 chimera capsid, each expressing a GFP transgene under thecontrol of the CAG promoter. FIG. 9A: Cells that were differentiatedinto muscle progenitor cells from a human pluripotent stem cell linewere infected with novel AAV variant LANKIQRTDA+V708I (SEQ IDNO:43).CAG.GFP, novel AAV variant LANKTTNKDA+V708I (SEQ IDNO:48).CAG.GFP, novel AAV variant AAV6/AAV5 chimera.CAG.GFP or wild typecontrol AAV9.CAG.GFP at an MOI of 500. Immunofluorescence imaging of thecell cultures 6 days after infection at all MOIs demonstrate that thenovel AAV variant capsids transduce muscle progenitor cells better thanwild type AAV9. FIG. 9B: Quantification of the percent of GFP-positivemuscle progenitor cells in each culture by flow cytometry reveals thatthe novel AAV variant capsids provide for a significant improvement inthe number of cells transduced over wild type AAV9. *p<0.05

FIGS. 10A-B provide data on the magnitude of improvement of transductionof human cardiomyocytes and human skeletal myofibers in vitro byrecombinant AAV virus comprising the novel AAV variant LANKIQRTDA+V708I(SEQ ID NO:43) capsid, the novel AAV variant LANKTTNKDA+V708I (SEQ IDNO:48) capsid, and the novel AAV variant AAV6/AAV5 chimera capsid, eachexpressing a GFP transgene under the control of the CAG promoter. FIG.10A: Fold increase in transduction of human cardiomyocytes by the novelAAV capsid variants compared to wild type AAV8 and AAV9, the serotypesmost widely used in clinical applications for muscle diseases. FIG. 10B:Fold increase in transduction of human skeletal myofibers by the novelAAV capsid variants compared to wild type AAV8 and AAV9.

FIGS. 11A-B provide data on the transduction of mouse tissue in vivo byrecombinant AAV virus comprising the novel AAV variant LANKIQRTDA+V708I(SEQ ID NO:43) capsid expressing a luciferase transgene under thecontrol of the CAG promoter. The mice were given a single intravenousinjection via the tail vein of 2×10″ viral genomes per animal. FIG. 11A:In life imaging of luciferase at day 14 (left) and day 28 (right)post-administration demonstrate that the novel AAV variantLANKIQRTDA+V708I (SEQ ID NO:43) capsid can transduce mouse cells invivo. FIG. 11B: Luciferase activity in heart, diaphragm, and quadriceps56 days post-administration demonstrate that the novel AAV variantLANKIQRTDA+V708I (SEQ ID NO:43) capsid can transduce mouse cardiac andskeletal muscle in vivo.

FIGS. 12A-B provide data on the transduction of non-human primateskeletal muscle in vivo by recombinant AAV virus comprising the novelAAV variant LANKIQRTDA+V708I (SEQ ID NO:43) capsid expressing a GFPtransgene under the control of the CAG promoter. The non-human primatewas given 3 intramuscular injections of 10¹¹ viral genomes each into theleft vastus lateralis, and the muscle tissue was analyzed 4 weekspost-administration. FIG. 12A: Representative images of haemotoxylin andeosin (H&E) and anti-GFP antibody staining of cross-sections of theproximal biopsy site at 2×, 4×, and 20× magnification demonstrate thatthe novel AAV variant LANKIQRTDA+V708I (SEQ ID NO:43) capsid cantransduce primate skeletal muscle cells in vivo. FIG. 12B:Representative images of haemotoxylin and eosin (H&E) and anti-GFPantibody staining of longitudinal sections of the distal biopsy site at2×, 4×, and 20× magnification demonstrate that the novel AAV variantLANKIQRTDA+V708I (SEQ ID NO:43) capsid can transduce primate skeletalmuscle cells in vivo.

DETAILED DESCRIPTION

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to a particular method orcomposition described and as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

The invention disclosed herein is illustrated in the figures anddescription. However, while particular embodiments are illustrated inthe figures, there is no intention to limit the invention to thespecific embodiment or embodiments illustrated and/or disclosed. Rather,the invention disclosed herein is intended to cover all modifications,alternative constructions, and equivalents falling within the spirit andscope of the invention. As such, the figures are intended to beillustrative and not restrictive.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It is noted that as used herein and in the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “arecombinant AAV virion” includes a plurality of such virions andreference to “the muscle cell” includes reference to one or more musclecells and equivalents thereof known to those skilled in the art, and soforth. It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

Adeno-associated virus is a nonpathogenic parvovirus composed of a 4.7kb single-stranded DNA genome within a non-enveloped, icosahedralcapsid. The genome contains three open reading frames (ORF) flanked byinverted terminal repeats (ITR) that function as the viral origin ofreplication and packaging signal. The rep ORF encodes four nonstructuralproteins that play roles in viral replication, transcriptionalregulation, site-specific integration, and virion assembly. The cap ORFencodes three structural proteins (VP 1-3) that assemble to form a60-mer viral capsid. Finally, an ORF present as an alternate readingframe within the cap gene produces the assembly-activating protein(AAP), a viral protein that localizes AAV capsid proteins to thenucleolus and functions in the capsid assembly process.

There are several naturally occurring (“wild-type”) serotypes and over100 known variants of AAV, each of which differs in amino acid sequence,particularly within the hypervariable regions of the capsid proteins,and thus in their gene delivery properties. No AAV has been associatedwith any human disease, making recombinant AAV attractive for clinicalapplications.

For the purposes of the disclosure herein, the terminology “AAV” is anabbreviation for adeno-associated virus, including, without limitation,the virus itself and derivatives thereof. Except where otherwiseindicated, the terminology refers to all subtypes or serotypes and bothreplication-competent and recombinant forms. The term “AAV” includes,without limitation, AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 orAAV2), AAV type 3A (AAV-3A or AAV3A), AAV type 3B (AAV-3B or AAV3B), AAVtype 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 orAAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type9 (AAV-9 or AAV9), AAV type 10 (AAV-10 or AAV10 or AAVrh10), avian AAV,bovine AAV, canine AAV, caprine AAV, equine AAV, primate AAV,non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infectprimates, “non-primate AAV” refers to AAV that infect non-primatemammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.

The genomic sequences of various serotypes of AAV, as well as thesequences of the native terminal repeats (TRs), Rep proteins, and capsidsubunits are known in the art. Such sequences may be found in theliterature or in public databases such as GenBank. See, e.g., GenBankAccession Numbers NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2(AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3A),NC_001729.1 (AAV3A), AF028705.1 (AAV3B), NC_001829.1 (AAV4), U89790.1(AAV4), NC_006152.1 (AA5), AF085716.1 (AAV-5), AF028704.1 (AAV6),NC_006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8) NC_006261.1(AAV-8), AY530579.1 (AAV9), AAT46337 (AAV10) and AA088208 (AAVrh10); thedisclosures of which are incorporated by reference herein for teachingAAV nucleic acid and amino acid sequences. See also, e.g., Srivistava etal. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology71:6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal etal. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994;Muramatsu et al. (1996) Virology 221:208; Shade et. al. (1986) J. Virol.58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris etal. (2004) Virology 33:375-383; international patent publications WO00/28061, WO 99/61601, WO 98/11244; and U.S. Pat. No. 6,156,303.

The sequences of naturally existing cap (capsid) proteins associatedwith AAV serotypes are known in the art and include those disclosedherein as AAV1 (SEQ ID NO:1), AAV2 (SEQ ID NO:2), AAV3A (SEQ ID NO:3),AAV3B (SEQ ID NO:4), AAV4 (SEQ ID NO:5), AAVS (SEQ ID NO:6), AAV6 (SEQID NO:7), AAV7 (SEQ ID NO:8), AAV8 (SEQ ID NO:9), AAV9 (SEQ ID NO:10),AAV10 (SEQ ID NO:11), and AAVrh10 (SEQ ID NO:12). The terms “variant AAVcapsid protein” or “AAV variant’ refer to an AAV capsid proteincomprising an amino acid sequence that includes at least onemodification or substitution (including deletion, insertion, pointmutation, etc.) relative to a naturally existing or “wild-type” AAVcapsid protein sequences, e.g. as set forth in SEQ ID NO:1-12 herein. Avariant AAV capsid protein may have about 80% identity or more to theamino acid sequence of a wild type capsid protein, for example, 85%identity or more, 90% identity or more, or 95% identity or more to theamino acid sequence of the wild type capsid protein, for example, 98% or99% identity to the wild type capsid protein. A variant AAV capsidprotein may not be a wild type capsid protein.

For the purposes of the disclosure herein, “AAV virion” or “AAV viralparticle” refers to a viral particle composed of at least one AAV capsidprotein and an encapsidated AAV polynucleotide.

For the purposes of the disclosure herein, the terminology “rAAV” is anabbreviation that refers to recombinant adeno-associated virus.“Recombinant,” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

The term “rAAV vector” encompasses rAAV virions (i.e., rAAV viralparticles) (e.g., an infectious rAAV virion), which by definitioninclude an rAAV polynucleotide; and also encompasses polynucleotidesencoding rAAV (e.g., a single stranded polynucleotide encoding rAAV(ss-rAAV); a double stranded polynucleotide encoding rAAV (ds-rAAV),e.g., plasmids encoding rAAV; and the like).

If an AAV virion comprises a heterologous polynucleotide (i.e. apolynucleotide other than a wild-type AAV genome, e.g., a transgene tobe delivered to a target cell, an RNAi agent or CRISPR agent to bedelivered to a target cell, etc.), it is typically referred to as a“recombinant AAV (rAAV) virion” or an “rAAV viral particle.” In general,the heterologous polynucleotide is flanked by at least one, andgenerally by two, AAV inverted terminal repeat sequences (ITRs).

The term “packaging” refers to a series of intracellular events thatresult in the assembly and encapsidation of an AAV particle. AAV “rep”and “cap” genes refer to polynucleotide sequences encoding replicationand encapsidation proteins of adeno-associated virus. AAV rep and capare referred to herein as AAV “packaging genes.”

The terminology “helper virus” for AAV refers to a virus that allows AAV(e.g. wild-type AAV) to be replicated and packaged by a mammalian cell.A variety of such helper viruses for AAV are known in the art, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC. Viruses of the herpesfamily include, for example, herpes simplex viruses (HSV) andEpstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) andpseudorabies viruses (PRV); which are also available from depositoriessuch as ATCC.

The terminology “helper virus function(s)” refers to function(s) encodedin a helper virus genome which allow AAV replication and packaging (inconjunction with other requirements for replication and packagingdescribed herein). As described herein, “helper virus function” may beprovided in a number of ways, including by providing helper virus orproviding, for example, polynucleotide sequences encoding the requisitefunction(s) to a producer cell in trans. For example, a plasmid or otherexpression vector comprising nucleotide sequences encoding one or moreadenoviral proteins is transfected into a producer cell along with anrAAV vector.

The terminology “infectious” virus or viral particle is one thatcomprises a competently assembled viral capsid and is capable ofdelivering a polynucleotide component into a cell for which the viralspecies is tropic. The term does not necessarily imply any replicationcapacity of the virus. Assays for counting infectious viral particlesare described elsewhere in this disclosure and in the art. Viralinfectivity can be expressed as the ratio of infectious viral particlesto total viral particles. Methods of determining the ratio of infectiousviral particle to total viral particle are known in the art. See, e.g.,Grainger et al. (2005) Mol. Ther. 11: S337 (describing a TCID50infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973.See also the Examples.

The term “tropism” as used herein refers to the preferential targetingby a virus (e.g., an AAV) of cells of a particular host species or ofparticular cell types within a host species. For example, a virus thatcan infect cells of the heart, lung, liver, and muscle has a broader(i.e., increased) tropism relative to a virus that can infect only lungand muscle cells. Tropism can also include the dependence of a virus onparticular types of cell surface molecules of the host. For example,some viruses can infect only cells with surface glycosaminoglycans,while other viruses can infect only cells with sialic acid (suchdependencies can be tested using various cells lines deficient inparticular classes of molecules as potential host cells for viralinfection). In some cases, the tropism of a virus describes the virus'srelative preferences. For example, a first virus may be able to infectall cell types but is much more successful in infecting those cells withsurface glycosaminoglycans. A second virus can be considered to have asimilar (or identical) tropism as the first virus if the second virusalso prefers the same characteristics (e.g., the second virus is alsomore successful in infecting those cells with surfaceglycosaminoglycans), even if the absolute transduction efficiencies arenot similar. For example, the second virus might be more efficient thanthe first virus at infecting every given cell type tested, but if therelative preferences are similar (or identical), the second virus canstill be considered to have a similar (or identical) tropism as thefirst virus. In some embodiments, the tropism of a virion comprising asubject variant AAV capsid protein is not altered relative to anaturally occurring virion. In some embodiments, the tropism of a virioncomprising a subject variant AAV capsid protein is expanded (i.e.,broadened) relative to a naturally occurring virion. In someembodiments, the tropism of a virion comprising a subject variant AAVcapsid protein is reduced relative to a naturally occurring virion.

The terminology “replication-competent” virus (e.g. areplication-competent AAV) refers to a phenotypically wild-type virusthat is infectious, and is also capable of being replicated in aninfected cell (i.e. in the presence of a helper virus or helper virusfunctions). In the case of AAV, replication competence generallyrequires the presence of functional AAV packaging genes. In general,rAAV vectors as described herein are replication-incompetent inmammalian cells (especially in human cells) by virtue of the lack of oneor more AAV packaging genes. Typically, such rAAV vectors lack any AAVpackaging gene sequences in order to minimize the possibility thatreplication competent AAV are generated by recombination between AAVpackaging genes and an incoming rAAV vector. In many embodiments, rAAVvector preparations as described herein are those which contain few ifany replication competent AAV (rcAAV, also referred to as RCA) (e.g.,less than about 1 rcAAV per 10² rAAV particles, less than about 1 rcAAVper 10⁴ rAAV particles, less than about 1 rcAAV per 10 rAAV particles,less than about 1 rcAAV per 10¹² rAAV particles, or no rcAAV).

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs, and may beinterrupted by non-nucleotide components. If present, modifications tothe nucleotide structure may be imparted before or after assembly of thepolymer. The term polynucleotide, as used herein, refers interchangeablyto double- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment herein that comprises a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same whencomparing the two sequences. Sequence similarity can be determined in anumber of different manners. To determine sequence identity, sequencescan be aligned using the methods and computer programs, including BLAST,available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Anotheralignment algorithm is FASTA, available in the Genetics Computing Group(GCG) package, from Madison, Wis., USA, a wholly owned subsidiary ofOxford Molecular Group, Inc. Other techniques for alignment aredescribed in Methods in Enzymology, vol. 266: Computer Methods forMacromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press,Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Ofparticular interest are alignment programs that permit gaps in thesequence. The Smith-Waterman is one type of algorithm that permits gapsin sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also,the GAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

The term “gene” refers to a polynucleotide that performs a function ofsome kind in the cell. For example, a gene can contain an open readingframe that is capable of encoding a gene product. One example of a geneproduct is a protein, which is transcribed and translated from the gene.Another example of a gene product is an RNA, e.g. a functional RNAproduct, e.g., an aptamer, an interfering RNA, a ribosomal RNA (rRNA), atransfer RNA (tRNA), a non-coding RNA (ncRNA), a guide RNA fornucleases, etc., which is transcribed but not translated.

The terminology “gene expression product” or “gene product” is amolecule resulting from expression of a particular gene, as definedabove. Gene expression products include, e.g., a polypeptide, anaptamer, an interfering RNA, a messenger RNA (mRNA), an rRNA, a tRNA, anon-coding RNA (ncRNA), and the like.

The term “siRNA agent” (“small interfering” or “short interfering RNA”(or siRNA)) is an RNA duplex of nucleotides that is targeted to a geneof interest (a “target gene”). An “RNA duplex” refers to the structureformed by the complementary pairing between two regions of a RNAmolecule, forming a region of double stranded RNA (dsRNA). siRNA is“targeted” to a gene in that the nucleotide sequence of the duplexportion of the siRNA is complementary to a nucleotide sequence of thetargeted gene. In some embodiments, the length of the duplex of siRNAsis less than 30 nucleotides. In some embodiments, the duplex can be 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11or 10 nucleotides in length. In some embodiments, the length of theduplex is 19-25 nucleotides in length. In some embodiments,siRNA-mediated gene targeting is accomplished through the use ofDNA-directed RNA interference (ddRNAi) which is a gene-silencingtechnique that utilizes DNA constructs to activate an animal cell'sendogenous RNA interference (RNAi) pathways. Such DNA constructs aredesigned to express self-complementary double-stranded RNAs, typicallyshort-hairpin RNAs (shRNA), that once processed bring about silencing ofa target gene or genes. Any RNA, including endogenous mRNAs or viralRNAs, can be silenced by designing constructs to express double-strandedRNA complementary to the desired mRNA target. As such, the RNA duplexportion of an siRNA agent can be part of a short hairpin structurereferred to as shRNA. In addition to the duplex portion, the hairpinstructure may contain a loop portion positioned between the twosequences that form the duplex. The loop can vary in length. In someembodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides inlength. The hairpin structure can also contain 3′ or 5′ overhangportions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0,1, 2, 3, 4 or 5 nucleotides in length. In general, the level ofexpression product (e.g., mRNA, polypeptide, etc.) of a target gene isreduced by an siRNA agent (e.g., an siRNA, an shRNA, etc.) that containsspecific double stranded nucleotide sequences that are complementary toat least a 19-25 nucleotide long segment (e.g., a 20-21 nucleotidesequence) of the target gene transcript, including the 5′ untranslated(UT) region, the ORF, or the 3′ UT region. In some embodiments, shortinterfering RNAs are about 19-25nt in length. See, e.g., PCTapplications WO 00/44895, WO 99/32619, WO 01/75164, WO 01/92513, WO01/29058, WO 01/89304, WO 02/16620, and WO 02/29858; and U.S. PatentPublication No. 2004/0023390 for descriptions of siRNA technology. ThesiRNA and/or shRNA can be encoded by a nucleic acid sequence, and thenucleic acid sequence can also include a promoter. The nucleic acidsequence can also include a polyadenylation signal. In some embodiments,the polyadenylation signal is a synthetic minimal polyadenylationsignal.

The terminology “antisense RNA” encompasses RNA that is complementary toa gene expression product. For example, an antisense RNA targeted to aspecific mRNA is an RNA-based agent (or can be a modified RNA) that iscomplementary to the mRNA, where hybridization of the antisense RNA tothe mRNA alters the expression of the mRNA (e.g., via altering thestability of the RNA, altering the translation of the RNA, etc.). Alsoincluded in “antisense RNA” are nucleic acids encoding an antisense RNA.

With regards to “CRISPR/Cas9 agents”, the term “CRISPR” encompassesClustered regularly interspaced short palindromicrepeats/CRISPR-associated (Cas) systems that evolved to provide bacteriaand archaea with adaptive immunity against viruses and plasmids by usingCRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids.The Cas9 protein (or functional equivalent and/or variant thereof, i.e.,Cas9-like protein) naturally contains DNA endonuclease activity thatdepends on association of the protein with two naturally occurring orsynthetic RNA molecules called crRNA and tracrRNA (also called guideRNAs). In some cases, the two molecules are covalently linked to form asingle molecule (also called a single guide RNA (“sgRNA”)). Thus, theCas9 or Cas9-like protein associates with a DNA-targeting RNA (whichterm encompasses both the two-molecule guide RNA configuration and thesingle-molecule guide RNA configuration), which activates the Cas9 orCas9-like protein and guides the protein to a target nucleic acidsequence.

If the Cas9 or Cas9-like protein retains its natural enzymatic function,it will cleave target DNA to create a double-strand break, which canlead to genome alteration (i.e., editing: deletion, insertion (when adonor polynucleotide is present), replacement, etc.), thereby alteringgene expression. Some variants of Cas9 (which variants are encompassedby the term Cas9-like) have been altered such that they have a decreasedDNA cleaving activity (in some cases, they cleave a single strandinstead of both strands of the target DNA, while in other cases, theyhave severely reduced to no DNA cleavage activity). Cas9-like proteinswith decreased DNA-cleavage activity (even no DNA-cleaving activity) canstill be guided to a target DNA to block RNA polymerase activity.Alternatively, the Cas9 or Cas9-like protein may be modified by fusing aVP64 transcription activation domain to the Cas9 protein andcodelivering the fusion protein with a MS2-P65-HSF1 helper protein and asingle guide RNA comprising MS2 RNA aptamers at the tetraloop andstem-loop to form a Synergistic Activation Mediator (Cas9-SAM) complexin the cell that activates transcription. Thus enzymatically inactiveCas9-like proteins can be targeted to a specific location in a targetDNA by a DNA-targeting RNA in order to block or activate transcriptionof the target DNA. The term “CRISPR/Cas9 agents” as used hereinencompasses all forms of CRISPR/Cas9 as described above or as known inthe art.

Detailed information regarding CRISPR agents can be found, for examplein (a) Jinek et. al., Science. 2012 Aug. 17; 337(6096):816-21: “Aprogrammable dual-RNA-guided DNA endonuclease in adaptive bacterialimmunity”; (b) Qi et al., Cell. 2013 Feb. 28; 152(5): 1173-83:“Repurposing CRISPR as an RNA-guided platform for sequence-specificcontrol of gene expression”, and (c) U.S. patent application Ser. No.13/842,859 and PCT application number PCT/US13/32589; all of which arehereby incorporated by reference in their entirety. Thus, the term“CRISPR agent” as used herein encompasses any agent (or nucleic acidencoding such an agent), comprising naturally occurring and/or syntheticsequences, that can be used in the Cas9-based system (e.g., a Cas9 orCas9-like protein; any component of a DNA-targeting RNA, e.g., acrRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donorpolynucleotide; and the like).

By “Zinc-finger nucleases” (ZFNs) it is meant artificial DNAendonucleases generated by fusing a zinc finger DNA binding domain to aDNA cleavage domain. ZFNs can be engineered to target desired DNAsequences and this enables zinc-finger nucleases to cleave unique targetsequences. When introduced into a cell, ZFNs can be used to edit targetDNA in the cell (e.g., the cell's genome) by inducing double strandbreaks. For more information on the use of ZFNs, see, for example: Asuriet al., Mol. Ther. 2012 February; 20(2):329-38; Bibikova et al. Science.2003 May 2; 300(5620):764; Wood et al. Science. 2011 Jul. 15;333(6040):307; Ochiai et al. Genes Cells. 2010 August; 15(8):875-85;Takasu et. al., Insect Biochem Mol Biol. 2010 October; 40(10):759-65;Ekker et al, Zebrafish 2008 Summer; 5(2): 121-3; Young et al, Proc NatlAcad Sci USA. 2011 Apr. 26; 108(17):7052-7; Goldberg et al, Cell. 2010Mar. 5; 140(5):678-91; Geurts et al, Science. 2009 Jul. 24;325(5939):433; Flisikowska et al, PLoS One. 2011; 6(6):e21045. doi:10.1371/journal.pone.0021045. Epub 2011 Jun. 13; Hauschild et al, ProcNatl Acad Sci USA. 2011 Jul. 19; 108(29): 12013-7; and Yu et al, CellRes. 2011 November; 21(1 1): 1638-40; all of which are hereinincorporated by reference for their teachings related to ZFNs. The term“ZFN agent” encompasses a zinc finger nuclease and/or a polynucleotidecomprising a nucleotide sequence encoding a zinc finger nuclease.

The terminology “Transcription activator-like effector nuclease” or“TALEN” agents refers to Transcription activator-like effector nucleases(TALENs). TALENs are artificial DNA endonucleases generated by fusing aTAL (Transcription activator-like) effector DNA binding domain to a DNAcleavage domain. TALENs can be quickly engineered to bind practicallyany desired DNA sequence and when introduced into a cell, TALENs can beused to edit target DNA in the cell (e.g., the cell's genome) byinducing double strand breaks. For more information on the use ofTALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul. 7;29(8):731-4; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Tesson etal. Nat Biotechnol. 2011 Aug. 5; 29(8):695-6; and Huang et. al., NatBiotechnol. 2011 Aug. 5; 29(8):699-700; all of which are hereinincorporated by reference for their teachings related to TALENs. Theterm “TALEN agent” encompasses a TALEN and/or a polynucleotidecomprising a nucleotide sequence encoding a TALEN.

The terminology “control element” or “control sequence” refers to anucleotide sequence involved in an interaction of molecules thatcontributes to the functional regulation of a polynucleotide, includingreplication, duplication, transcription, splicing, translation, ordegradation of the polynucleotide. The regulation may affect thefrequency, speed, or specificity of the process, and may be enhancing orinhibitory in nature. Control elements known in the art include, forexample, transcriptional regulatory sequences such as promoters andenhancers. A promoter is a DNA region capable under certain conditionsof binding RNA polymerase and initiating transcription of a codingregion usually located downstream (in the 3′ direction) from thepromoter. Promoters may be ubiquitously acting, i.e. active in many celltypes, e.g. CAG or CMV promoters; or tissue or cell specific, e.g. thepromoter can be tissue-specific for expression in cardiomyocytes.

The terminology “operatively linked” or “operably linked” refers to ajuxtaposition of genetic elements, wherein the elements are in arelationship permitting them to operate in the expected manner. Forinstance, a promoter is operatively linked to a coding region if thepromoter helps initiate transcription of the coding sequence. There maybe intervening residues between the promoter and coding region so longas this functional relationship is maintained.

The terminology “expression vector” encompasses a vector comprising apolynucleotide region which encodes a polypeptide of interest, and isused for effecting the expression of the protein in an intended targetcell. An expression vector may also comprise control elementsoperatively linked to the encoding region to facilitate expression ofthe protein in the target. The combination of control elements and agene or genes to which they are operably linked for expression issometimes referred to as an “expression cassette,” a large number ofwhich are known and available in the art or can be readily constructedfrom components that are available in the art.

The term “heterologous” means derived from a genotypically distinctentity from that of the rest of the entity to which it is beingcompared. For example, a polynucleotide introduced by geneticengineering techniques into a plasmid or vector derived from a differentspecies is a heterologous polynucleotide. A promoter removed from itsnative coding sequence and operatively linked to a coding sequence withwhich it is not naturally found linked is a heterologous promoter. Thus,for example, an rAAV that includes a heterologous nucleic acid sequenceencoding a heterologous gene product is an rAAV that includes apolynucleotide not normally included in a naturally-occurring, wild-typeAAV, and the encoded heterologous gene product is a gene product notnormally encoded by a naturally-occurring, wild type AAV.

The terminology “genetic alteration” and “genetic modification” (andgrammatical variants thereof), are used interchangeably herein to referto a process wherein a genetic element (e.g., a polynucleotide) isintroduced into a cell other than by mitosis or meiosis. The element maybe heterologous to the cell, or it may be an additional copy or improvedversion of an element already present in the cell. Genetic alterationmay be effected, for example, by transfecting a cell with a recombinantplasmid or other polynucleotide through any process known in the art,such as electroporation, calcium phosphate precipitation, or contactingwith a polynucleotide-liposome complex. Genetic alteration may also beeffected, for example, by transduction or infection with a DNA or RNAvirus or viral vector. Generally, the genetic element is introduced intoa chromosome or mini-chromosome in the cell; but any alteration thatchanges the phenotype and/or genotype of the cell and its progeny isincluded in this term.

With regards to cell modification, the terminology “geneticallymodified” or “transformed” or “transfected” or “transduced” by exogenousDNA (e.g. via a recombinant virus) refers to when such DNA has beenintroduced inside the cell. The presence of the exogenous DNA results inpermanent or transient genetic change. The transforming DNA may or maynot be integrated (covalently linked) into the genome of the cell. A“clone” is a population of cells derived from a single cell or commonancestor by mitosis. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

As used herein, a cell is said to be “stably” altered, transduced,genetically modified, or transformed with a genetic sequence if thesequence is available to perform its function during extended culture ofthe cell in vitro and/or for an extended period of time in vivo.Generally, such a cell is “heritably” altered (genetically modified) inthat a genetic alteration is introduced which is also inheritable byprogeny of the altered cell.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The terms also encompass an amino acid polymer that has beenmodified; for example, disulfide bond formation, glycosylation,lipidation, phosphorylation, or conjugation with a labeling component.Polypeptides such as anti-angiogenic polypeptides, neuroprotectivepolypeptides, and the like, when discussed in the context of deliveringa gene product to a mammalian subject, and compositions therefor, referto the respective intact polypeptide, or any fragment or geneticallyengineered derivative thereof, which retains the desired biochemicalfunction of the intact protein. Similarly, references to nucleic acidsencoding anti-angiogenic polypeptides, nucleic acids encodingneuroprotective polypeptides, and other such nucleic acids for use indelivery of a gene product to a mammalian subject (which may be referredto as “transgenes” to be delivered to a recipient cell), includepolynucleotides encoding the intact polypeptide or any fragment orgenetically engineered derivative possessing the desired biochemicalfunction.

As used herein, an “isolated” plasmid, nucleic acid, vector, virus,virion, host cell, protein, or other substance refers to a preparationof the substance devoid of at least some of the other components thatmay also be present where the substance or a similar substance naturallyoccurs or is initially prepared from. Thus, for example, an isolatedsubstance may be prepared by using a purification technique to enrich itfrom a source mixture. Enrichment can be measured on an absolute basis,such as weight per volume of solution, or it can be measured in relationto a second, potentially interfering substance present in the sourcemixture. Increasing enrichments of the embodiments of this disclosureare increasingly more isolated. An isolated plasmid, nucleic acid,vector, virus, host cell, or other substance is in some embodimentspurified, e.g., from about 80% to about 90% pure, at least about 90%pure, at least about 95% pure, at least about 98% pure, or at leastabout 99%, or more, pure.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease (and/or symptoms caused by thedisease) from occurring in a subject which may be predisposed to thedisease or at risk of acquiring the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease (and/or symptomscaused by the disease), i.e., arresting its development; and (c)relieving the disease (and/or symptoms caused by the disease), i.e.,causing regression of the disease (and/or symptoms caused by thedisease), i.e., ameliorating the disease and/or one or more symptoms ofthe disease. For example, the subject compositions and methods may bedirected towards the treatment of muscle disease. Nonlimiting methodsfor assessing muscle diseases and the treatment thereof includemeasuring therapeutic protein production (e.g. muscle biopsy followed byimmunohistochemistry or serum sampling followed by ELISA or enzymeactivity assays), measuring symptoms of heart failure (e.g. the New YorkHeart Association Functional Classification or the Minnesota Living WithHeart Failure Questionnaire), functional cardiac status (e.g. the6-minute walk test or peak maximum oxygen consumption), biomarkeranalysis (e.g. N-terminal prohormone brain natriuretic peptide), leftventricular function/remodeling (e.g. left ventricular ejection fractionor left ventricular end-systolic volume), muscle strength (e.g. theMedical Research Council Scales Clinical Investigation of DuchenneDystrophy, hand-held dynamometry, or maximum weight lift), musclefunction (e.g. the Vignos Scale, Timed Function Tests, the HammersmithMotor Ability Score, timed rise from floor, walk tests, Motor FunctionMeasure Scale, North Star Ambulatory Assessment, 9 Hole Peg Test, orChildren's Hospital of Philadelphia Infant Test of NeuromuscularDisorders), muscle disease symptoms (e.g. the Neuromuscular SymptomsScore or Clinical Global Impressions), mitochondrial function (e.g. ³¹Pmagnetic resonance spectroscopy), questionnaire-based assessments ofquality of life, patient-reported outcomes, or daily activities.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, humans; non-human primates, including simians; mammaliansport animals (e.g., horses); mammalian farm animals (e.g., sheep,goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g.,mice, rats, etc.).

In some embodiments, the individual is a human who has previously beennaturally exposed to AAV and as a result harbors anti-AAV antibodies(i.e., AAV neutralizing antibodies). In some embodiments, the individualis a human who has previously been administered an AAV vector (and as aresult may harbor anti-AAV antibodies) and needs re-administration ofvector for treatment of a different condition or for further treatmentof the same condition. Based on positive results in clinical trialsinvolving AAV gene delivery to, for example, liver, muscle, andretina—all tissues affected by neutralizing antibodies against thisvehicle—there are many such therapeutic applications/disease targets.

The term “effective amount” as used herein is an amount sufficient toeffect beneficial or desired clinical results. An effective amount canbe administered in one or more administrations. For purposes of thisdisclosure, an effective amount of a compound (e.g., an infectious rAAVvirion) is an amount that is sufficient to palliate, ameliorate,stabilize, reverse, prevent, slow or delay the progression of (and/orsymptoms associated with) a particular disease state (e.g., a muscledisease). Accordingly, an effective amount of an infectious rAAV virionis an amount of the infectious rAAV virion that is able to effectivelydeliver a heterologous nucleic acid to a target cell (or target cells)of the individual. Effective amounts may be determined preclinically by,e.g., detecting in the cell or tissue the gene product (RNA, protein)that is encoded by the heterologous nucleic acid sequence usingtechniques that are well understood in the art, e.g. RT-PCR, westernblotting, ELISA, fluorescence or other reporter readouts, and the like.Effective amounts may be determined clinically by, e.g. detecting achange in the onset or progression of disease using methods known in theart, e.g. 6-minute walk test, left ventricular ejection fraction,hand-held dynamometry, Vignos Scale and the like as described herein andas known in the art.

The terminology “muscle cell” or “muscle tissue” refers herein to a cellor group of cells derived from muscle of any kind, including, withoutlimitation, skeletal muscle, cardiac muscle, smooth muscle (e.g. fromthe digestive tract, urinary bladder and blood vessels) and diaphragmmuscle. Such muscle cells may be differentiated or undifferentiated suchas myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.Since muscle tissue is readily accessible to the circulatory system, aprotein produced and secreted by muscle cells and tissue in vivo willlogically enter the bloodstream for systemic benefit, thereby providingsustained, therapeutic levels of protein secretion from the muscle.

The terminology “directed evolution” refers to a capsid engineeringmethodology, in vitro and/or in vivo, which emulates natural evolutionthrough iterative rounds of genetic diversification and selectionprocesses, thereby accumulating beneficial mutations that progressivelyimprove the function of a biomolecule. Directed evolution often involvesan in vivo method referred to as “biopanning” for selection of AAVvariants from a library which variants possess a more efficient level ofinfectivity of a cell or tissue type of interest.

DETAILED DESCRIPTION

Adeno-associated viruses (AAVs) are a family of parvoviruses with a 4.7kb single-stranded DNA genome contained inside a non-enveloped capsid.The viral genome of a naturally occurring AAV has 2 inverted terminalrepeats (ITR)—which function as the viral origin of replication andpackaging signal—flanking 2 primary open reading frames (ORF): rep(encoding proteins that function in viral replication, transcriptionalregulation, site-specific integration, and virion assembly) and cap. Thecap ORF codes for 3 structural proteins that assemble to form a 60-merviral capsid. Many naturally occurring AAV variants and serotypes havebeen isolated, and none have been associated with human disease.

Recombinant versions of AAV can be used as gene delivery vectors, wherea marker or therapeutic gene of interest is inserted between the ITRs inplace of rep and cap. These vectors have been shown to transduce bothdividing and non-dividing cells in vitro and in vivo and can result instable transgene expression for years in post-mitotic tissue. See e.g.,Knipe D M, Howley P M. Fields' Virology. Lippincott Williams & Wilkins,Philadelphia, Pa., USA, 2007; Gao G-P, Alvira M R, Wang L, Calcedo R,Johnston J, Wilson J M. Novel adeno-associated viruses from rhesusmonkeys as vectors for human gene therapy. Proc Natl Acad Sci USA 2002;99: 11854-9; Atchison R W, Casto B C, Hammon W M. Adenovirus-AssociatedDefective Virus Particles. Science 1965; 149: 754-6; Hoggan M D,Blacklow N R, Rowe W P. Studies of small DNA viruses found in variousadenovirus preparations: physical, biological, and immunologicalcharacteristics. Proc Natl Acad Sci USA 1966; 55: 1467-74; Blacklow N R,Hoggan M D, Rowe W P. Isolation of adenovirus-associated viruses fromman. Proc Natl Acad Sci USA 1967; 58: 1410-5; Bantel-Schaal U, zurHausen H. Characterization of the DNA of a defective human parvovirusisolated from a genital site. Virology 1984; 134: 52-63; Mayor H D,Melnick J L. Small deoxyribonucleic acid-containing viruses(picodnavirus group). Nature 1966; 210: 331-2; Mori S, Wang L, TakeuchiT, Kanda T. Two novel adeno-associated viruses from cynomolgus monkey:pseudotyping characterization of capsid protein. Virology 2004; 330:375-83; Flotte T R. Gene therapy progress and prospects: recombinantadeno-associated virus (rAAV) vectors. Gene Ther 2004; 11: 805-10.

Recombinant AAV (referred to herein simply as “AAV”) has yieldedpromising results in an increasing number of clinical trials. However,there are impediments to gene delivery that may limit AAV's utility,such as anti-capsid immune responses, low transduction of certaintissues, an inability for targeted delivery to specific cell types and arelatively low carrying capacity. In many situations, there isinsufficient mechanistic knowledge to effectively empower rationaldesign with the capacity to improve AAV. As an alternative, directedevolution has emerged as a strategy to create novel AAV variants thatmeet specific biomedical needs. Directed evolution strategies harnessgenetic diversification and selection processes to enable theaccumulation of beneficial mutations that progressively improve thefunction of a biomolecule. In this process, wild-type AAV cap genes arediversified by several approaches to create large genetic libraries thatare packaged to generate libraries of viral particles, and selectivepressure is then applied to isolate novel variants that can overcomegene delivery barriers. Importantly, the mechanistic basis underlying agene delivery problem does not need to be known for directed evolutionof function, which can thus accelerate the development of enhancedvectors.

Typically, the variants disclosed herein were generated through use ofan AAV library and/or libraries. Such an AAV library or libraries is/aregenerated by mutating the cap gene, the gene which encodes thestructural proteins of the AAV capsid, by a range of directed evolutiontechniques known by and readily available to the skilled artisan in thefield of viral genome engineering. See e.g., Bartel et al. Am. Soc. GeneCell Ther. 15^(th) Annu. Meet. 20, S140 (2012); Bowles, D. et al. J.Virol. 77, 423-432 (2003); Gray et al. Mol. Ther. 18, 570-578 (2010);Grimm, D. et al. J. Virol. 82, 5887-5911; Koerber, J. T. et al. Mol.Ther. 16, 1703-1709 (2008); Li W. et al. Mol. Ther. 16, 1252-1260(2008); Koerber, J. T. et al. Methods Mol. Biol. 434, 161-170 (2008);Koerber, J. T. et al. Hum. Gene Ther. 18, 367-378 (2007); and Koerber,J. T. et al. Mol. Ther. 17, 2088-2095 (2009). Such techniques, withoutlimitation, are as follows: i) Error-prone PCR to introduce random pointmutations into the AAV cap open reading frame (ORF) at a predetermined,modifiable rate; ii) In vitro or in vivo viral recombination or “DNAshuffling” to generate random chimeras of AAV cap genes to yield a genelibrary with multiple AAV serotypes; iii) Random peptide insertions atdefined sites of the capsid by ligation of degenerate oligonucleotidesin the cap ORF; iv) Defined insertions of peptide-encoding sequencesinto random locations of the AAV cap ORF using transposon mutagenesis;v) Replacing surface loops of AAV capsids with libraries of peptidesequences bioinformationally designed based on the level of conservationof each amino acid position among natural AAV serotypes and variants togenerate “loop-swap” libraries; vi) Random amino acid substitution atpositions of degeneracy between AAV serotypes to generate libraries ofancestral variants (Santiago-Ortiz et al., 2015); and a combination ofsuch techniques thereof.

DNA shuffling generates chimeras which combine their parental propertiesin unique and, often beneficial, ways; however, some may be incapable ofpackaging which, in effect, reduces the diversity of the library.Concentration of diversity the library into specific region(s) of thecapsid is achieved through peptide insertion techniques such as, withoutlimitation, iii-iv) above. Diversity of the library is also concentratedinto specific region(s) of the capsid in techniques such as v) above,and such concentration is directed onto multiple hypervariable regions,which lie on surface exposed loops, of the AAV capsid. While many of thetechniques generate variant capsids with only a small area of the capsidmutated, these techniques can be paired with additional mutagenesisstrategies to modify the full capsid.

Once the AAV library or libraries is/are generated, viruses are thenpackaged, such that each AAV particle is comprised of a mutant capsidsurrounding a cap gene encoding that capsid, and purified. Variants ofthe library are then subjected to in vitro and/or in vivo selectivepressure techniques known by and readily available to the skilledartisan in the field of AAV. See e.g., Maheshri, N. et al. NatureBiotech. 24, 198-204 (2006); Dalkara, D. et al. Sci. Transl. Med. 5,189ra76 (2013); Lisowski, L. et al. Nature. 506, 382-286 (2013); Yang,L. et al. PNAS. 106, 3946-3951 (2009); Gao, G. et al. Mol. Ther. 13,77-87 (2006); and Bell, P. et al. Hum. Gene. Ther. 22, 985-997 (2011).For example, without limitation, AAV variants can be selected using i)affinity columns in which elution of different fractions yields variantswith altered binding properties; ii) primary cells—isolated from tissuesamples or immortal cell lines that mimic the behavior of cells in thehuman body—which yield AAV variants with increased efficiency and/ortissue specificity; iii) animal models—which mimic a clinical genetherapy environment—which yield AAV variants that have successfullyinfected target tissue; iv) human xenograft models which yield AAVvariants that have infected grafted human cells; and/or a combination ofselection techniques thereof.

Once viruses are selected, they may be recovered by known techniquessuch as, without limitation, adenovirus-mediated replication, PCRamplification, Next Generation sequencing and cloning, and the like.Virus clones are then enriched through repeated rounds of the selectiontechniques and AAV DNA is isolated to recover selected variant cap genesof interest. Such selected variants can be subjected to furthermodification or mutation and as such serve as a new starting point forfurther selection steps to iteratively increase AAV viral fitness.However, in certain instances, successful capsids have been generatedwithout additional mutation.

The AAV variants disclosed herein were generated at least in partthrough the use of in vivo directed evolution methodology, such as thetechniques described above, involving the use of primate cardiac andskeletal muscle screens following intravenous administration. As such,the AAV variant capsids disclosed herein comprise one or moremodifications in amino acid sequence that confer more efficienttransduction of primate muscle cells than a corresponding parental AAVcapsid protein. As used herein, a “corresponding parental AAV capsidprotein” refers to an AAV capsid protein of the same wild-type orvariant AAV serotype as the subject variant AAV capsid protein but thatdoes not comprise the one or more amino acid sequence modifications ofthe subject variant AAV capsid protein. In particular embodiments, anAAV comprising a variant AAV capsid protein as herein described hassystemic tropism toward cardiac muscle and/or multiple skeletal musclegroups throughout the body following systemic or tissue-targetedadministration.

In some embodiments, the subject variant AAV capsid protein comprises aheterologous peptide of from about 5 amino acids to about 20 amino acidsinserted by covalent linkage into an AAV capsid protein GH loop, or loopIV, relative to a corresponding parental AAV capsid protein. By the “GHloop,” or loop IV, of the AAV capsid protein it is meant thesolvent-accessible portion referred to in the art as the GH loop, orloop IV, of AAV capsid protein. For the GH loop/loop IV of AAV capsid,see, e.g., van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al.(2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955.Thus, for example, the insertion site can be within about amino acids411-650 of an AAV VP1 capsid protein. For example, the insertion sitecan be within amino acids 571-612 of AAV1 VP1, amino acids 570-611 ofAAV2 VP1, within amino acids 571-612 of AAV3A VP1, within amino acids571-612 of AAV3B VP1, within amino acids 569-610 of AAV4 VP1, withinamino acids 560-601 of AAV5 VP1, within amino acids 571 to 612 of AAV6VP1, within amino acids 572 to 613 of AAV7 VP1, within amino acids 573to 614 of AAV8 VP1, within amino acids 571 to 612 of AAV9 VP1, or withinamino acids 573 to 614 of AAV10 VP1, or the corresponding amino acids ofany variant thereof. Those skilled in the art would know, based on acomparison of the amino acid sequences of capsid proteins of various AAVserotypes, where an insertion site “corresponding to amino acids ofAAV2” would be in a capsid protein of any given AAV serotype. See alsoFIG. 6 for an alignment of wild-type AAV SEQ ID NOS:1-11 which providesamino acid locations between and across the wild-type (naturallyoccurring) serotypes AAV1, AAV2, AAV3A, AAV3B, and AAV4-10.

In certain embodiments, the insertion site is a single insertion sitebetween two adjacent amino acids located between amino acids 570-614 ofVP1 of any wild-type AAV serotype or AAV variant, e.g., the insertionsite is between two adjacent amino acids located in amino acids 570-610,amino acids 580-600, amino acids 570-575, amino acids 575-580, aminoacids 580-585, amino acids 585-590, amino acids 590-600, or amino acids600-614, of VP1 of any AAV serotype or variant. For example, theinsertion site can be between amino acids 580 and 581, amino acids 581and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids585 and 586, amino acids 586 and 587, amino acids 587 and 588, aminoacids 588 and 589, or amino acids 589 and 590. The insertion site can bebetween amino acids 575 and 576, amino acids 576 and 577, amino acids577 and 578, amino acids 578 and 579, or amino acids 579 and 580. Theinsertion site can be between amino acids 590 and 591, amino acids 591and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids594 and 595, amino acids 595 and 596, amino acids 596 and 597, aminoacids 597 and 598, amino acids 598 and 599, or amino acids 599 and 600.For example, the insertion site can be between amino acids 587 and 588of AAV2, between amino acids 590 and 591 of AAV1, between amino acids588 and 589 of AAV3A, between amino acids 588 and 589 of AAV3B, betweenamino acids 584 and 585 of AAV4, between amino acids 575 and 576 ofAAV5, between amino acids 590 and 591 of AAV6, between amino acids 589and 590 of AAV7, between amino acids 590 and 591 of AAV8, between aminoacids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.

In some embodiments, a peptide insertion disclosed herein has a lengthof 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 aminoacids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids,14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 aminoacids, 19 amino acids, or 20 amino acids. In another embodiment, apeptide insertion disclosed herein comprises from 1 to 4 spacer aminoacids at the amino terminus (N-terminus) and/or at the carboxyl terminus(C-terminus) of any one of the peptide insertions disclosed herein.Exemplary spacer amino acids include, without limitation, leucine (L),alanine (A), glycine (G), serine (S), threonine (T), and proline (P). Incertain embodiments, a peptide insertion comprises 2 spacer amino acidsat the N-terminus and 2 spacer amino acids at the C-terminus. In otherembodiments, a peptide insertion comprises 2 spacer amino acids at theN-terminus and 1 spacer amino acids at the C-terminus.

The peptide insertions disclosed herein have not been previouslydescribed and/or inserted into an AAV capsid. Without wishing to bebound by theory, the presence of any of the disclosed peptide insertionsmay act to lower the variant capsid's affinity for heparin sulfate whichcould alter extracellular or intracellular steps within the viraltransduction pathway. In addition, the peptide insertion motifsdisclosed herein may confer enhanced transduction of muscle cells (e.g.cardiomyocytes) through the addition of a cell surface receptor bindingdomain.

In some preferred embodiments, the insertion peptide comprises an aminoacid sequence of any one of the formulas below.

In some aspects, an insertion peptide can be a peptide of 7 to 10 aminoacids in length, of Formula 1a:Y₁Y₂X₁X₂X₃X₄X₅X₆X₇Y₃

-   -   Where each of Y₁-Y₃, if present, is independently selected from        Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from Ala, Asn, Thr, Gly, Ser, Ala, Gln, and Asp    -   X₂ is selected from Lys, Asn, Thr, Ser, Ala, and Gln    -   X₃ is selected from Ile, Thr, Lys, Leu, Val, Asn, Asp, and Arg    -   X₄ is selected from Gln, Thr, Ile, Lys, Val, Ser, and Tyr    -   X₅ is selected from Arg, Asn, Gly, Lys, Leu, Thr, Ala, Ser, and        Gln    -   X₆ is selected from Thr, Lys, Val, Gly, Ser, Ala, Arg, and Pro    -   X₇ is selected from Asp, Thr, Asn, Ile, Ala, and Ser.

In certain embodiments, the insertion peptide of Formula 1a comprises anamino acid sequence selected from NKIQRTD (SEQ ID NO:13), NKTTNKD (SEQID NO:14), TNKIGVT (SEQ ID NO:15), GNLTKGN (SEQ ID NO:16), NTVKLST (SEQID NO:17), SNTVKAI (SEQ ID NO:18), ASNITKA (SEQ ID NO:19), DNTVTRS (SEQID NO:20), NKISAKD (SEQ ID NO:21), NQDYTKT (SEQ ID NO:22), QADTTKN (SEQID NO:23), TNRTSPD (SEQ ID NO:24), SNTTQKT (SEQ ID NO:25) and ASDSTKA(SEQ ID NO:26). In other embodiments, the insertion peptide of Formula1a does not comprise an amino acid sequence selected from NKTTNKD (SEQID NO:14), QADTTKN (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24) and NQDYTKT(SEQ ID NO:22).

In other aspects, an insertion peptide can be a peptide of 7 to 10 aminoa acids in length, of Formula 1b:Y₁Y₂X₁X₂X₃X₄X₅X₆X₇Y₃

-   -   Where each of Y₁-Y₃, if present, is independently selected from        Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from Thr and Asn    -   X₂ is selected from Asn and Lys    -   X₃ is selected from Lys, Ile and Thr    -   X₄ is selected from Ile, Gln, and Thr    -   X₅ is selected from Gly, Arg and Asn    -   X₆ is selected from Val, Thr and Lys    -   X₇ is selected from Thr and Asp

In certain embodiments, the insertion peptide of Formula 1b comprises anamino acid sequence selected from NKIQRTD (SEQ ID NO:13), NKTTNKD (SEQID NO:14) and TNKIGVT (SEQ ID NO:15). In other embodiments, theinsertion peptide of Formula 1a does not comprise the amino acidsequence NKTTNKD (SEQ ID NO:14).

In other aspects, an insertion peptide can be a peptide of 7 to 10 aminoacids in length, of Formula 1cY₁Y₂X₁X₂X₃X₄X₅X₆X₇Y₃

-   -   Where each of Y₁-Y₃, if present, is independently selected from        Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from Thr and Asn    -   X₂ is selected from Asn and Lys    -   X₃ is selected from Lys and Ile    -   X₄ is selected from Ile and Gln    -   X₅ is selected from Gly and Arg    -   X₆ is selected from Val and Thr    -   X₇ is selected from Thr and Asp

In certain embodiments, the insertion peptide of Formula 1c comprises anamino acid sequence selected from NKIQRTD (SEQ ID NO:13) and TNKIGVT(SEQ ID NO:15).

In other aspects, an insertion peptide can be a peptide of 7 to 10 aminoacids in length, of Formula 1d:Y₁Y₂X₁X₂X₃X₄X₅X₆X₇Y₃

-   -   Where each of Y₁-Y₃, if present, is independently selected from        Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from Asn and Thr    -   X₂ is selected from Asn and Lys    -   X₃ is selected from Lys and Thr    -   X₄ is selected from Ile and Thr    -   X₅ is selected from Gly, Lys and Thr    -   X₆ is selected from Lys, Arg and Val    -   X₇ is selected from Asp, Thr and Asn

In certain embodiments, the insertion peptide of Formula 1d comprisesthe amino acid sequence TNKIGVT (SEQ ID NO:15).

In other embodiments, the insertion peptide comprises an amino acidsequence selected from NKIQRTD (SEQ ID NO:13), NKTTNKD (SEQ ID NO:14)and TNKIGVT (SEQ ID NO:15). In related embodiments, the insertionpeptide comprises an amino acid sequence selected from NKIQRTD (SEQ IDNO:13) and TNKIGVT (SEQ ID NO:15).

In some embodiments, the insertion peptide comprises an amino acidsequence selected from NKIQRTD (SEQ ID NO:13), NKTTNKD (SEQ ID NO:14),TNKIGVT (SEQ ID NO:15), GNLTKGN (SEQ ID NO:16), NTVKLST (SEQ ID NO:17),SNTVKAI (SEQ ID NO:18), ASNITKA (SEQ ID NO:19), DNTVTRS (SEQ ID NO:20),NKISAKD (SEQ ID NO:21), NQDYTKT (SEQ ID NO:22), QADTTKN (SEQ ID NO:23),TNRTSPD (SEQ ID NO:24), SNTTQKT (SEQ ID NO:25) and ASDSTKA (SEQ IDNO:26).

In other preferred embodiments, the insertion peptide has from 1 to 3spacer amino acids (Y₁-Y₃) at the amino and/or carboxyl terminus of anamino acid sequence selected from NKIQRTD (SEQ ID NO:13), NKTTNKD (SEQID NO:14), TNKIGVT (SEQ ID NO:15), GNLTKGN (SEQ ID NO:16), NTVKLST (SEQID NO:17), SNTVKAI (SEQ ID NO:18), ASNITKA (SEQ ID NO:19), DNTVTRS (SEQID NO:20), NKISAKD (SEQ ID NO:21), NQDYTKT (SEQ ID NO:22), QADTTKN (SEQID NO:23), TNRTSPD (SEQ ID NO:24), SNTTQKT (SEQ ID NO:25) and ASDSTKA(SEQ ID NO:26). In certain such embodiments, the insertion peptide isselected from the group consisting of: LANKIQRTDA (SEQ ID NO:27),LANKTTNKDA (SEQ ID NO:28), LATNKIGVTA (SEQ ID NO:29), LAGNLTKGNA (SEQ IDNO:30), LANTVKLSTA (SEQ ID NO:31), LASNTVKAIA (SEQ ID NO:32), LAASNITKAA(SEQ ID NO:33), LADNTVTRSA (SEQ ID NO:34), LANKISAKDA (SEQ ID NO:35),LANQDYTKTA (SEQ ID NO:36), LATNKIGVTS (SEQ ID NO:37), LATNKIGVTA (SEQ IDNO:38), LAQADTTKNA (SEQ ID NO:39), LATNRTSPDA (SEQ ID NO:40), LASNTTQKTA(SEQ ID NO:41), and LAASDSTKAA (SEQ ID NO:42).

In some embodiments, the subject variant AAV capsid protein does notinclude any other amino acid sequence modifications other than a peptideinsertion of from about 5 amino acids to about 20 amino acids in the GHloop, or loop IV. For example, in some embodiments, the subject variantAAV capsid protein comprises a peptide insertion comprising an aminoacid sequence selected from the group consisting of NKIQRTD (SEQ IDNO:13), NKTTNKD (SEQ ID NO:14), TNKIGVT (SEQ ID NO:15), GNLTKGN (SEQ IDNO:16), NTVKLST (SEQ ID NO:17), SNTVKAI (SEQ ID NO:18), ASNITKA (SEQ IDNO:19), DNTVTRS (SEQ ID NO:20), NKISAKD (SEQ ID NO:21), NQDYTKT (SEQ IDNO:22), QADTTKN (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), SNTTQKT (SEQ IDNO:25), ASDSTKA (SEQ ID NO:26), LANKIQRTDA (SEQ ID NO:27), LANKTTNKDA(SEQ ID NO:28), LATNKIGVTA (SEQ ID NO:29), LAGNLTKGNA (SEQ ID NO:30),LANTVKLSTA (SEQ ID NO:31), LASNTVKAIA (SEQ ID NO:32), LAASNITKAA (SEQ IDNO:33), LADNTVTRSA (SEQ ID NO:34), LANKISAKDA (SEQ ID NO:35), LANQDYTKTA(SEQ ID NO:36), LATNKIGVTS (SEQ ID NO:37), LATNKIGVTA (SEQ ID NO:38),LAQADTTKNA (SEQ ID NO:39), LATNRTSPDA (SEQ ID NO:40), LASNTTQKTA (SEQ IDNO:41), and LAASDSTKAA (SEQ ID NO:42), and the variant AAV capsid doesnot include any other amino acid substitutions, insertions, or deletions(i.e., the variant AAV capsid protein comprises said insertion and isotherwise identical to the corresponding AAV capsid protein). Putanother way, the variant AAV capsid protein comprising said insertion isotherwise identical to the parental AAV capsid protein into which thepeptide has been inserted. As another example, the subject variant AAVcapsid protein comprises a peptide insertion comprising an amino acidsequence selected from NKIQRTD (SEQ ID NO:13), NKTTNKD (SEQ ID NO:14),TNKIGVT (SEQ ID NO:15), GNLTKGN (SEQ ID NO:16), NTVKLST (SEQ ID NO:17),SNTVKAI (SEQ ID NO:18), ASNITKA (SEQ ID NO:19), DNTVTRS (SEQ ID NO:20),NKISAKD (SEQ ID NO:21), NQDYTKT (SEQ ID NO:22), QADTTKN (SEQ ID NO:23),TNRTSPD (SEQ ID NO:24), SNTTQKT (SEQ ID NO:25), ASDSTKA (SEQ ID NO:26),LANKIQRTDA (SEQ ID NO:27), LANKTTNKDA (SEQ ID NO:28), LATNKIGVTA (SEQ IDNO:29), LAGNLTKGNA (SEQ ID NO:30), LANTVKLSTA (SEQ ID NO:31), LASNTVKAIA(SEQ ID NO:32), LAASNITKAA (SEQ ID NO:33), LADNTVTRSA (SEQ ID NO:34),LANKISAKDA (SEQ ID NO:35), LANQDYTKTA (SEQ ID NO:36), LATNKIGVTS (SEQ IDNO:37), LATNKIGVTA (SEQ ID NO:38), LAQADTTKNA (SEQ ID NO:39), LATNRTSPDA(SEQ ID NO:40), LASNTTQKTA (SEQ ID NO:41), and LAASDSTKAA (SEQ IDNO:42), wherein the peptide insertion is located between amino acids 587and 588 of the VP1 of the AAV2 capsid; between amino acids 588 and 589of VP1 of AAV3A, AAV3B, AAV9, or AAV10; between amino acids 589 and 590of VP1 of AAV7; between amino acids 590 to 591 of VP1 of AAV1, AAV6, orAAV8, between amino acids 584 and 585 of VP1 of AAV4, or between aminoacids 575 and 576 of AAV5, wherein the variant AAV capsid proteinsequence is otherwise identical to the corresponding parental AAV capsidprotein sequence, e.g. any one of SEQ ID NOs:1-12.

In other embodiments, the subject variant AAV capsid protein, inaddition to comprising a peptide insertion, e.g. as disclosed herein oras known in the art, in the GH loop, comprises from about 1 to about 100amino acid substitutions or deletions, e.g. 1 to about 5, from about 2to about 4, from about 2 to about 5, from about 5 to about 10, fromabout 10 to about 15, from about 15 to about 20, from about 20 to about25, from about 25-50, from about 50-100 amino acid substitutions ordeletions compared to the parental AAV capsid protein. Thus, in someembodiments, a subject variant capsid protein comprises an amino acidsequence having a sequence identity of 85% or more, 90% or more, 95% ormore, or 98% or more, e.g. or 99% identity to the corresponding parentalAAV capsid, e.g. a wild type capsid protein as set forth in SEQ IDNOs:1-12.

In a further embodiment, the one or more amino acid substitutions are atamino acid residue(s) 35, 109, 195, 213, 222, 229, 312, 319, 330, 333,347, 363, 427, 447, 449, 453, 490, 527, 551, 581, 585, 588, 593, 606,649, 651, 694, 698, 708, and/or 735 of AAV2 VP1 capsid protein asnumbered prior to insertion of the peptide, or the corresponding aminoacid residue(s) of another AAV capsid protein. In some such embodiments,the one or more amino acid substitutions are selected from the groupconsisting of A35P, S109T, P195L, D213N, G222S, V229I, N312K, A319T,T330A, A333S, E347K, P363L, A427D, V447F, N449D, N449K, G453R, A490T,K527Q, N551S, A581T, Y585S, R588M, A593E, W606C, K649E, R651H, W694C,I698V, V708I, and L735Q of AAV2 VP1 capsid protein as numbered prior tothe insertion of the peptide, or the corresponding amino acid residue(s)of another AAV capsid protein.

In a preferred embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom NKIQRTD (SEQ ID NO:13), NKTTNKD (SEQ ID NO:14) and TNKIGVT (SEQ IDNO:15), and b) one or more of the following amino acid substitutionscompared to the amino acid sequence of AAV2 (SEQ ID NO:2) or thecorresponding substitution in another AAV parental serotype (i.e. otherthan AAV2), wherein the substituted amino acid(s) do not naturally occurat the corresponding positions: A35P, S109T, P195L, D213N, G222S, V229I,N312K, A319T, T330A, A333S, E347K, P363L, A427D, V447F, N449D, N449K,G453R, A490T, K527Q, N551S, A581T, Y₅₈₅S, R588M, A593E, W606C, K649E,R651H, W694C, I698V, V708I, L735Q and a combination thereof. In someembodiments, the one or more amino acid substitutions are selected fromthe group consisting of: V708I, V708I+A593E, V708I+S109T, V708I+T330A,A35P, V708I+R588M, V708I+W606C, V708I+W694C, I698V,N312K+N449D+N551S+1698V+L735Q, N312K+N449D+N551S+1698V+V708I+L735Q,V708I+N449K, and V708I+G222S. Preferably, the peptide insertion site islocated between amino acids 587 and 588 of AAV2 capsid, between aminoacids 587 and 588 of AAV2 capsid, between amino acids 588 and 589 ofAAV3A, AAV3B, AAV9, or AAV10 capsid, between amino acids 589 and 590 ofAAV7 capsid, between amino acids 590 to 591 of AAV1, AAV6, or AAV8capsid, between amino acids 584 and 585 of AAV4 capsid, or between aminoacids 575 and 576 of AAV5 capsid.

In a particularly preferred embodiment, the variant AAV capsid comprisesa peptide insertion comprising the amino acid sequence NKIQRTD (SEQ IDNO:13) or comprising, consisting essentially of, or consisting of theamino acid sequence LANKIQRTDA (SEQ ID NO:27) between amino acids 587and 588 of VP1 of AAV2 or the corresponding amino acids of another AAVcapsid, and further comprises a V708I amino acid substitution at residue708 relative to the amino acid sequence of AAV2 capsid (SEQ ID NO:2) andoptionally further comprises an A593E and/or S109T and/or T330A and/orR588M substitution relative to AAV2 or the corresponding substitutionsin another AAV parental serotype, wherein the substituted amino acid(s)do not naturally occur at the corresponding position. In anotherparticularly preferred embodiment, the variant AAV capsid comprises apeptide insertion comprising the amino acid sequence NKIQRTD (SEQ IDNO:13) or comprising, consisting essentially of, or consisting of theamino acid sequence LANKIQRTDA (SEQ ID NO:27) between amino acids 587and 588 of VP1 of AAV2 or the corresponding amino acids of another AAVcapsid, and further comprises an A35P amino acid substitution at residue35 relative to the amino acid sequence of AAV2 capsid (SEQ ID NO:2) orthe corresponding substitution in another AAV parental serotype. Thevariant AAV capsid may have at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, or greater,amino acid sequence identity to the entire length of the amino acidsequence set forth in SEQ ID NO:2 or the corresponding parental AAVcapsid. In a particularly preferred embodiment, the variant AAV capsidhas an amino acid sequence having at least about 85%, at least about90%, at least about 95%, at least about 98% sequence identity to or is100% identical to the following amino acid sequence:

(SEQ ID NO: 43) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANKIQRTDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNY NKS INVDFTVDTNGVYSEPRPIGTRYLTRNL

In another particularly preferred embodiment, the variant AAV capsidcomprises a peptide insertion comprising the amino acid sequence NKIQRTD(SEQ ID NO:13) or comprising, consisting essentially of, or consistingof the amino acid sequence LANKIQRTDA (SEQ ID NO:27) between amino acids587 and 588 of AAV2 capsid protein or the corresponding position in thecapsid protein of another AAV serotype and comprises an N312K amino acidsubstitution compared to the amino acid sequence of AAV2 capsid (SEQ IDNO:2) or the corresponding substitution in another AAV parental serotypeand optionally further comprises (i) N449D, N551S, I698V and L735Q or(ii) N449D, N551S, I698V, L735Q and V708I amino acid substitutionscompared to the amino acid sequence of AAV2 capsid or the correspondingsubstitutions in another AAV parental serotype. The variant AAV capsidmay have at least about 85%, at least about 90%, at least about 95%, atleast about 98%, or greater, amino acid sequence identity to the entirelength of the amino acid sequence set forth in SEQ ID NO:2. In aparticularly preferred embodiment, the variant AAV capsid has an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 98% sequence identity to or is 100% identicalto the following amino acid sequence:

(SEQ ID NO: 44) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGF RPKRL KFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT D TPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT S VDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANKIQRTDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPE V QYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRN Q

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion located between amino acids 588 and589 of VP1 of AAV3A, AAV3B, AAV9, or AAV10, between amino acids 589 and590 of AAV7, between amino acids 590 to 591 of AAV1, AAV6 or AAV8,between amino acids 584 and 585 of AAV4 or between amino acids 575 and576 of AAV5, the peptide insertion comprising an amino acid sequenceselected from NKIQRTD (SEQ ID NO:13) and LANKIQRTDA (SEQ ID NO:27), andb) a valine to isoleucine substitution at amino acid 709 of AAV3A orAAV3B, an alanine to isoleucine substitution at position 709 of AAV1 orAAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4or amino acid 709 of AAV9 or a threonine to isoleucine substitution atamino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10 or a glutamineto isoleucine substitution at amino acid 697 of AAV5 and is optionallyotherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferredembodiments, the variant capsid protein comprises a) a peptide insertioncomprising the amino acid sequence NKIQRTD (SEQ ID NO:13) or comprising,consisting essentially of, or consisting of the amino acid sequenceLANKIQRTDA (SEQ ID NO:27) between amino acids 587 and 588 of AAV2 capsidand b) a valine to isoleucine amino acid substitution at amino acid 708compared to the amino acid sequence of AAV2, wherein the variant capsidprotein comprises from 2 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions.

In yet another embodiment, the variant capsid protein comprises a) apeptide insertion comprising the amino acid sequence NKIQRTD (SEQ IDNO:13) or comprising, consisting essentially of, or consisting of theamino acid sequence LANKIQRTDA (SEQ ID NO:27) between amino acids 587and 588 of AAV2 capsid and b) a valine to isoleucine amino acidsubstitution at amino acid 708 compared to the amino acid sequence ofAAV2 and is otherwise identical to the amino acid sequence of SEQ IDNO:2.

In yet another embodiment, the variant capsid protein comprises a) apeptide insertion comprising the amino acid sequence NKIQRTD (SEQ IDNO:13) or comprising, consisting essentially of, or consisting of theamino acid sequence LANKIQRTDA (SEQ ID NO:27) between amino acids 587and 588 of AAV2 capsid and is otherwise identical to the amino acidsequence of SEQ ID NO:2. In some embodiments, the variant AAV capsid hasan amino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98% sequence identity to or is 100%identical to the following amino acid sequence:

(SEQ ID NO: 45) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANKIQRTDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another particularly preferred embodiment, the variant AAV capsidcomprises a peptide insertion comprising the amino acid sequence TNKIGVT(SEQ ID NO:15) or comprising, consisting essentially of, or consistingof the amino acid sequence LATNKIGVTA (SEQ ID NO:29) or LATNKIGVTS (SEQID NO:37) between amino acids 587 and 588 of AAV2 capsid or thecorresponding position in the capsid protein of another AAV serotype andcomprises a V708I amino acid substitution compared to the amino acidsequence of AAV2 or the corresponding substitution in another AAVparental serotype and optionally further comprises an N449K and/or G222Ssubstitution relative to AAV2 or the corresponding substitution in thecapsid protein of another AAV parental serotype, wherein the substitutedamino acids do not naturally occur at the corresponding position. Inanother preferred embodiment, the variant AAV capsid comprises a peptideinsertion comprising the amino acid sequence TNKIGVT (SEQ ID NO:15) orcomprising, consisting essentially of, or consisting of the amino acidsequence LATNKIGVTA (SEQ ID NO:29) or LATNKIGVTS (SEQ ID NO:37) betweenamino acids 587 and 588 of AAV2 capsid or the corresponding position inthe capsid protein of another AAV serotype and comprises N312K, N449D,N551S, I698V and L735Q and optionally V708I amino acid substitutionscompared to the amino acid sequence of AAV2 or the correspondingsubstitution(s) in another AAV parental serotype, wherein thesubstituted amino acid(s) do not naturally occur at the correspondingposition. The variant AAV capsid may have at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or greater, aminoacid sequence identity to the entire length of the amino acid sequenceset forth in SEQ ID NO:2. In a particularly preferred embodiment, thevariant AAV capsid has an amino acid sequence having at least about 85%,at least about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 46) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTEEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LATNKIGVTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNY NKS INVDFTVDTNGVYSEPRPIGTRYLTRNL

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion located between amino acids 588 and589 of VP1 of AAV3A, AAV3B, AAV9, or AAV10, between amino acids 589 and590 of AAV7, between amino acids 590 to 591 of AAV1, AAV6 or AAV8,between amino acids 584 and 585 of AAV4 or between amino acids 575 and576 of AAV5, the peptide insertion comprising an amino acid sequenceselected from TNKIGVT (SEQ ID NO:15), LATNKIGVTA (SEQ ID NO:29) andLATNKIGVTS (SEQ ID NO:37), and b) a valine to isoleucine substitution atamino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitutionat position 709 of AAV1 or AAV6, an asparagine to isoleucinesubstitution at amino acid 707 of AAV4 or amino acid 709 of AAV9 or athreonine to isoleucine substitution at amino acid 710 of AAV7 or aminoacid 711 of AAV8 or AAV10 or a glutamine to isoleucine substitution atamino acid 697 of AAV5. In preferred embodiments, the variant AAV capsidcomprises a peptide insertion comprising the amino acid sequence TNKIGVT(SEQ ID NO:15) or comprising, consisting essentially of, or consistingof the amino acid sequence LATNKIGVTA (SEQ ID NO:29) or LATNKIGVTS (SEQID NO:37) between amino acids 587 and 588 of AAV2 capsid and comprises avaline to isoleucine amino acid substitution at amino acid 708 (V708I)compared to the amino acid sequence of AAV2, wherein the variant capsidprotein comprises from 2 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions and is preferably at least about 85%, at least about 90%,at least about 95%, at least about 98%, or greater amino acid sequenceidentity to the entire length of the amino acid sequence set forth inSEQ ID NO:2.

In yet another embodiment, the variant capsid protein comprises a) apeptide insertion comprising the amino acid sequence TNKIGVT (SEQ IDNO:15) or comprising, consisting essentially of, or consisting of theamino acid sequence LATNKIGVTA (SEQ ID NO:29) or LATNKIGVTS (SEQ IDNO:37) between amino acids 587 and 588 of AAV2 capsid and b) a valine toisoleucine amino acid substitution at amino acid 708 compared to theamino acid sequence of AAV2 and is otherwise identical to the amino acidsequence of SEQ ID NO:2.

In yet another embodiment, the variant capsid protein comprises a) apeptide insertion comprising the amino acid sequence TNKIGVT (SEQ IDNO:15) or comprising, consisting essentially of, or consisting of theamino acid sequence LATNKIGVTA (SEQ ID NO:29) or LATNKIGVTS (SEQ IDNO:37) between amino acids 587 and 588 of AAV2 capsid and is otherwiseidentical to the amino acid sequence of SEQ ID NO:2. In someembodiments, the variant AAV capsid has an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about98% sequence identity to or is 100% identical to the following aminoacid sequence:

(SEQ ID NO: 47) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LATNKIGVTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another preferred embodiment, the variant AAV capsid comprises apeptide insertion comprising the amino acid sequence NKTTNKD (SEQ IDNO:14) or LANKTTNKDA (SEQ ID NO:28) between amino acids 587 and 588 ofAAV2 capsid and further comprises a V708I amino acid substitution atresidue 708 relative to the amino acid sequence of AAV2 capsid (SEQ IDNO:2) or the corresponding substitution in another AAV parental serotypeand optionally further comprises an S109T and/or W694C and/or W606Camino acid substitution compared to the amino acid sequence of AAV2 orthe corresponding substitution in another AAV parental serotype, whereinthe substituted amino acid(s) do not naturally occur at thecorresponding position. In another particularly preferred embodiment,the variant AAV capsid comprises a peptide insertion comprising theamino acid sequence NKTTNKD (SEQ ID NO:14) or comprising, consistingessentially of, or consisting of the amino acid sequence LANKTTNKDA (SEQID NO:28) between amino acids 587 and 588 of VP1 of AAV2 or thecorresponding amino acids of another AAV capsid, and further comprisesan I698V amino acid substitution at residue 698 relative to the aminoacid sequence of AAV2 capsid (SEQ ID NO:2) or the corresponding residueof another AAV capsid. The variant AAV capsid may have at least about85%, at least about 90%, at least about 95%, at least about 98%, orgreater amino acid sequence identity to the entire length of the aminoacid sequence set forth in SEQ ID NO:2 or the corresponding parental AAVcapsid. In a particularly preferred embodiment, the variant AAV capsidhas an amino acid sequence having at least about 85%, at least about90%, at least about 95%, at least about 98% sequence identity to or is100% identical to the following amino acid sequence:

(SEQ ID NO: 48) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANKTTNKDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSN YNKS INVDFTVDTNGVYSEPRPIGTRYLTRNL

In another particularly preferred embodiment, the variant AAV capsidcomprises a peptide insertion comprising the amino acid sequence NKTTNKD(SEQ ID NO:14) or comprising, consisting essentially of, or consistingof the amino acid sequence LANKTTNKDA (SEQ ID NO:28) between amino acids587 and 588 of AAV2 capsid protein or the corresponding position in thecapsid protein of another AAV serotype and comprises an N312K amino acidsubstitution compared to the amino acid sequence of AAV2 capsid (SEQ IDNO:2) or the corresponding substitution in another AAV parental serotypeand optionally further comprises N449D, N551S, I698V, and L735Q andoptionally V708I amino acid substitutions compared to the amino acidsequence of AAV2 capsid or the corresponding substitutions in anotherAAV parental serotype. The variant AAV capsid may have at least about85%, at least about 90%, at least about 95%, at least about 98%, orgreater, amino acid sequence identity to the entire length of the aminoacid sequence set forth in SEQ ID NO:2. In a particularly preferredembodiment, the variant AAV capsid has an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about98% sequence identity to or is 100% identical to the following aminoacid sequence:

(SEQ ID NO: 49) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGF RPKRL KFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT D TPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT S VDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANKTTNKDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPE V QYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRN Q

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion located between amino acids 588 and589 of VP1 of AAV3A, AAV3B, AAV9, or AAV10, between amino acids 589 and590 of AAV7, between amino acids 590 to 591 of AAV1, AAV6 or AAV8,between amino acids 584 and 585 of AAV4 or between amino acids 575 and576 of AAV5, the peptide insertion comprising an amino acid sequenceselected from NKTTNKD (SEQ ID NO:14) and LANKTTNKDA (SEQ ID NO:28), andb) a valine to isoleucine substitution at amino acid 709 of AAV3A orAAV3B, an alanine to isoleucine substitution at position 709 of AAV1 orAAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4or amino acid 709 of AAV9 or a threonine to isoleucine substitution atamino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10 or a glutamineto isoleucine substitution at amino acid 697 of AAV5 and is optionallyotherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferredembodiments, the variant capsid protein comprises a) a peptide insertioncomprising the amino acid sequence NKTTNKD (SEQ ID NO:14) or comprising,consisting essentially of, or consisting of the amino acid sequenceLANKTTNKDA (SEQ ID NO:28) between amino acids 587 and 588 of AAV2 capsidand b) a valine to isoleucine amino acid substitution at amino acid 708compared to the amino acid sequence of AAV2, wherein the variant capsidprotein comprises from 2 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions.

In yet another embodiment, the variant capsid protein comprises a) apeptide insertion comprising the amino acid sequence NKTTNKD (SEQ IDNO:14) or comprising, consisting essentially of, or consisting of theamino acid sequence LANKTTNKDA (SEQ ID NO:28) between amino acids 587and 588 of AAV2 capsid and b) a valine to isoleucine amino acidsubstitution at amino acid 708 compared to the amino acid sequence ofAAV2 and is otherwise identical to the amino acid sequence of SEQ IDNO:2.

In another embodiment, the variant capsid comprises a peptide insertioncomprising the amino acid sequence NKTTNKD (SEQ ID NO:14) or comprising,consisting essentially of, or consisting of the amino acid sequenceLANKTTNKDA (SEQ ID NO:28) between amino acids 587 and 588 of AAV2 capsidand is otherwise identical to the amino acid sequence set forth in SEQID NO:2. In some embodiments, the variant AAV capsid has an amino acidsequence having at least about 85%, at least about 90%, at least about95%, at least about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 50) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANKTTNKDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL 

In other embodiments, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom GNLTKGN (SEQ ID NO:16), NTVKLST (SEQ ID NO:17), SNTVKAI (SEQ IDNO:18), ASNITKA (SEQ ID NO:19), DNTVTRS (SEQ ID NO:20), NKISAKD (SEQ IDNO:21), NQDYTKT (SEQ ID NO:22), QADTTKN (SEQ ID NO:23), TNRTSPD (SEQ IDNO:24), SNTTQKT (SEQ ID NO:25) and ASDSTKA (SEQ ID NO:26), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: A35P, S109T, P195L, D213N, G222S, V229I, N312K, A319T, T330A,A333S, E347K, P363L, A427D, V447F, N449D, N449K, G453R, A490T, K527Q,N551S, A581T, Y585S, R588M, A593E, W606C, K649E, R651H, W694C, I698V,V708I, L735Q and a combination thereof. In some embodiments, the one ormore amino acid substitutions are selected from the group consisting of:V708I, S109T, R651H, A319T, P195L, P363L, I698V, D213N, G453R and acombination thereof. In some preferred embodiments, the one or moreamino acid substitutions include at least a V708I and/or P363L aminoacid substitution or the corresponding substitution in another AAVparental serotype. Preferably, the peptide insertion site is locatedbetween amino acids 587 and 588 of AAV2 capsid or the correspondingposition in the capsid protein of another AAV serotype.

In some embodiments, the variant AAV capsid comprises a peptideinsertion comprising the amino acid sequence GNLTKGN (SEQ ID NO:16) orcomprising, consisting essentially of, or consisting of the amino acidsequence LAGNLTKGNA (SEQ ID NO:30) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid andfurther comprises one or more of the following amino acid substitutionsrelative to the amino acid sequence of AAV2 capsid (SEQ ID NO:2) or thecorresponding substitution in another AAV parental serotype, wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: V708I, V708I+S109T, R651H, A319T+P195L, P363L, P363L+V708I.In some embodiments, the variant AAV capsid comprises (i) a peptideinsertion comprising the amino acid sequence GNLTKGN (SEQ ID NO:16) orcomprising, consisting essentially of, or consisting of the amino acidsequence LAGNLTKGNA (SEQ ID NO:30) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid and(ii) a V708I substitution relative to the amino acid sequence of AAV2capsid (SEQ ID NO:2) or the corresponding residue of another AAV capsidand comprises from 2 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions or is otherwise identical to the amino acid sequence ofSEQ ID NO:2 or the to the corresponding parental AAV capsid proteinsequence. In other embodiments, the variant AAV capsid comprises (i) apeptide insertion comprising the amino acid sequence GNLTKGN (SEQ IDNO:16) or comprising, consisting essentially of, or consisting of theamino acid sequence LAGNLTKGNA (SEQ ID NO:30) between amino acids 587and 588 of VP1 of AAV2 or the corresponding amino acids of another AAVcapsid and (ii) a P363L substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding residue of another AAVcapsid and comprises from 2 to 5, from 5 to 10, or from 10 to 15 aminoacid substitutions or is otherwise identical to the amino acid sequenceof SEQ ID NO:2 or the to the corresponding parental AAV capsid proteinsequence. In other embodiments, the variant AAV capsid comprises (i) apeptide insertion comprising the amino acid sequence GNLTKGN (SEQ IDNO:16) or comprising, consisting essentially of, or consisting of theamino acid sequence LAGNLTKGNA (SEQ ID NO:30) between amino acids 587and 588 of VP1 of AAV2 or the corresponding amino acids of another AAVcapsid and (ii) an R651H substitution relative to the amino acidsequence of AAV2 capsid (SEQ ID NO:2) or the corresponding residue ofanother AAV capsid and comprises from 2 to 5, from 5 to 10, or from 10to 15 amino acid substitutions or is otherwise identical to the aminoacid sequence of SEQ ID NO:2 or the to the corresponding parental AAVcapsid protein sequence. In another embodiment, the variant capsidcomprises a peptide insertion comprising the amino acid sequence GNLTKGN(SEQ ID NO:16) or comprising, consisting essentially of, or consistingof the amino acid sequence LAGNLTKGNA (SEQ ID NO:30) between amino acids587 and 588 of AAV2 capsid and is otherwise identical to the amino acidsequence set forth in SEQ ID NO:2. In some embodiments, the variant AAVcapsid has an amino acid sequence having at least about 85%, at leastabout 90%, at least about 95%, at least about 98% sequence identity toor is 100% identical to the following amino acid sequence:

(SEQ ID NO: 51) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAGNLTKGNA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In some embodiments, the variant AAV capsid comprises (i) a peptideinsertion comprising the amino acid sequence NTVKLST (SEQ ID NO:17) orcomprising, consisting essentially of, or consisting of the amino acidsequence LANTVKLSTA (SEQ ID NO:31) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid and(ii) a V708I amino acid substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) the corresponding substitution in anotherAAV parental serotype, wherein the substituted amino acid(s) do notnaturally occur at the corresponding positions, and comprises from 2 to5, from 5 to 10, or from 10 to 15 amino acid substitutions or isotherwise identical to the amino acid sequence of SEQ ID NO:2 or to thecorresponding parental AAV capsid protein sequence. In anotherembodiment, the variant capsid comprises a peptide insertion comprisingthe amino acid sequence NTVKLST (SEQ ID NO:17) or comprising, consistingessentially of, or consisting of the amino acid sequence LANTVKLSTA (SEQID NO:31) between amino acids 587 and 588 of AAV2 capsid and isotherwise identical to the amino acid sequence set forth in SEQ ID NO:2.In some embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 52) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTEEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANTVKLSTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In some embodiments, the variant AAV capsid comprises (i) a peptideinsertion comprising the amino acid sequence SNTVKAI (SEQ ID NO:18) orcomprising, consisting essentially of, or consisting of the amino acidsequence LASNTVKAIA (SEQ ID NO:32) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid and(ii) a V708I amino acid substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype, and comprises from 2 to 5, from 5 to 10,or from 10 to 15 amino acid substitutions or is otherwise identical tothe amino acid sequence of SEQ ID NO:2 or the to the correspondingparental AAV capsid protein sequence. In another embodiment, the variantcapsid comprises a peptide insertion comprising the amino acid sequenceSNTVKAI (SEQ ID NO:18) or comprising, consisting essentially of, orconsisting of the amino acid sequence LASNTVKAIA (SEQ ID NO:32) betweenamino acids 587 and 588 of AAV2 capsid and is otherwise identical to theamino acid sequence set forth in SEQ ID NO:2. In some embodiments, thevariant AAV capsid has an amino acid sequence having at least about 85%,at least about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 53) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTEEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LASNTVKAIA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In some embodiments, the variant AAV capsid comprises (i) a peptideinsertion comprising the amino acid sequence ASNITKA (SEQ ID NO:19) orcomprising, consisting essentially of, or consisting of the amino acidsequence LAASNITKAA (SEQ ID NO:33) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid and(ii) a V708I amino acid substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype, and comprises from 2 to 5, from 5 to 10,or from 10 to 15 amino acid substitutions or is otherwise identical tothe amino acid sequence of SEQ ID NO:2 or the to the correspondingparental AAV capsid protein sequence. In another embodiment, the variantcapsid comprises a peptide insertion comprising the amino acid sequenceASNITKA (SEQ ID NO:19) or comprising, consisting essentially of, orconsisting of the amino acid sequence LAASNITKAA (SEQ ID NO:33) betweenamino acids 587 and 588 of AAV2 capsid and is otherwise identical to theamino acid sequence set forth in SEQ ID NO:2. In some embodiments, thevariant AAV capsid has an amino acid sequence having at least about 85%,at least about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 54) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTEEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAASNITKAA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In some embodiments, the variant AAV capsid comprises (i) a peptideinsertion comprising the amino acid sequence DNTVTRS (SEQ ID NO:20) orcomprising, consisting essentially of, or consisting of the amino acidsequence LADNTVTRSA (SEQ ID NO:34) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid and(ii) a V708I amino acid substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype, and comprises from 2 to 5, from 5 to 10,or from 10 to 15 amino acid substitutions or is otherwise identical tothe amino acid sequence of SEQ ID NO:2 or the to the correspondingparental AAV capsid protein sequence. In other embodiments, the variantAAV capsid comprises (i) a peptide insertion comprising the amino acidsequence DNTVTRS (SEQ ID NO:20) or comprising, consisting essentiallyof, or consisting of the amino acid sequence LADNTVTRSA (SEQ ID NO:34)between amino acids 587 and 588 of VP1 of AAV2 or the correspondingamino acids of another AAV capsid and (ii) an I698V amino acidsubstitution relative to the amino acid sequence of AAV2 capsid (SEQ IDNO:2) or the corresponding substitution in another AAV parentalserotype, wherein the substituted amino acid does not naturally occur atthe corresponding position, and comprises from 2 to 5, from 5 to 10, orfrom 10 to 15 amino acid substitutions or is otherwise identical to theamino acid sequence of SEQ ID NO:2 or the to the corresponding parentalAAV capsid protein sequence. In another embodiment, the variant capsidcomprises a peptide insertion comprising the amino acid sequence DNTVTRS(SEQ ID NO:20) or comprising, consisting essentially of, or consistingof the amino acid sequence LADNTVTRSA (SEQ ID NO:34) between amino acids587 and 588 of AAV2 capsid and is otherwise identical to the amino acidsequence set forth in SEQ ID NO:2. In some embodiments, the variant AAVcapsid has an amino acid sequence having at least about 85%, at leastabout 90%, at least about 95%, at least about 98% sequence identity toor is 100% identical to the following amino acid sequence:

(SEQ ID NO: 55) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTEEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LADNTVTRSA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In some embodiments, the variant AAV capsid comprises (i) a peptideinsertion comprising the amino acid sequence NKISAKD (SEQ ID NO:21) orcomprising, consisting essentially of, or consisting of the amino acidsequence LANKISAKDA (SEQ ID NO:35) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid and(ii) a V708I amino acid substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype, and comprises from 2 to 5, from 5 to 10,or from 10 to 15 amino acid substitutions or is otherwise identical tothe amino acid sequence of SEQ ID NO:2 or the to the correspondingparental AAV capsid protein sequence. In another embodiment, the variantcapsid comprises a peptide insertion comprising the amino acid sequenceNKISAKD (SEQ ID NO:21) or comprising, consisting essentially of, orconsisting of the amino acid sequence LANKISAKDA (SEQ ID NO:35) betweenamino acids 587 and 588 of AAV2 capsid and is otherwise identical to theamino acid sequence set forth in SEQ ID NO:2. In some embodiments, thevariant AAV capsid has an amino acid sequence having at least about 85%,at least about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 56) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANKISAKDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In some embodiments, the variant AAV capsid comprises (i) a peptideinsertion comprising the amino acid sequence NQDYTKT (SEQ ID NO:22) orcomprising, consisting essentially of, or consisting of the amino acidsequence LANQDYTKTA (SEQ ID NO:36) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid and(ii) a V708I amino acid substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype and comprises from 2 to 5, from 5 to 10,or from 10 to 15 amino acid substitutions or is otherwise identical tothe amino acid sequence of SEQ ID NO:2 or the to the correspondingparental AAV capsid protein sequence. In other embodiments, the variantAAV capsid comprises (i) a peptide insertion comprising the amino acidsequence NQDYTKT (SEQ ID NO:22) or comprising, consisting essentiallyof, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO:36)between amino acids 587 and 588 of VP1 of AAV2 or the correspondingamino acids of another AAV capsid and (ii) an I698V amino acidsubstitution relative to the amino acid sequence of AAV2 capsid (SEQ IDNO:2) or the corresponding substitution in another AAV parental serotype(i.e. other than AAV2), wherein the substituted amino acid does notnaturally occur at the corresponding position, and comprises from 2 to5, from 5 to 10, or from 10 to 15 amino acid substitutions or isotherwise identical to the amino acid sequence of SEQ ID NO:2 or the tothe corresponding parental AAV capsid protein sequence. In anotherembodiment, the variant capsid comprises a peptide insertion comprisingthe amino acid sequence NQDYTKT (SEQ ID NO:22) or comprising, consistingessentially of, or consisting of the amino acid sequence LANQDYTKTA (SEQID NO:36) between amino acids 587 and 588 of AAV2 capsid and isotherwise identical to the amino acid sequence set forth in SEQ ID NO:2.In some embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 57) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTEEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANQDYTKTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In some embodiments, the variant AAV capsid comprises a peptideinsertion comprising the amino acid sequence QADTTKN (SEQ ID NO:23) orcomprising, consisting essentially of, or consisting of the amino acidsequence LAQADTTKNA (SEQ ID NO:39) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid andfurther comprises one or more of the following amino acid substitutionsrelative to the amino acid sequence of AAV2 capsid (SEQ ID NO:2) or thecorresponding substitutions in another AAV parental serotype, whereinthe substituted amino acid(s) do not naturally occur at thecorresponding positions: V708I, D213N, P363L, G453R. In someembodiments, the variant AAV capsid comprises (i) a peptide insertioncomprising the amino acid sequence QADTTKN (SEQ ID NO:23) or comprising,consisting essentially of, or consisting of the amino acid sequenceLAQADTTKNA (SEQ ID NO:39) between amino acids 587 and 588 of VP1 of AAV2or the corresponding amino acids of another AAV capsid and (ii) a V708Isubstitution relative to the amino acid sequence of AAV2 capsid (SEQ IDNO:2) or the corresponding substitution in another AAV parental serotypeand comprises from 2 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions or is otherwise identical to the amino acid sequence ofSEQ ID NO:2 or the to the corresponding parental AAV capsid proteinsequence. In other embodiments, the variant AAV capsid comprises (i) apeptide insertion comprising the amino acid sequence QADTTKN (SEQ IDNO:23) or comprising, consisting essentially of, or consisting of theamino acid sequence LAQADTTKNA (SEQ ID NO:39) between amino acids 587and 588 of VP1 of AAV2 or the corresponding amino acids of another AAVcapsid and (ii) a P363L substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding residue of another AAVcapsid and comprises from 2 to 5, from 5 to 10, or from 10 to 15 aminoacid substitutions or is otherwise identical to the amino acid sequenceof SEQ ID NO:2 or the to the corresponding parental AAV capsid proteinsequence. In other embodiments, the variant AAV capsid comprises (i) apeptide insertion comprising the amino acid sequence QADTTKN (SEQ IDNO:23) or comprising, consisting essentially of, or consisting of theamino acid sequence LAQADTTKNA (SEQ ID NO:39) between amino acids 587and 588 of VP1 of AAV2 or the corresponding amino acids of another AAVcapsid and (ii) a D213N substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype, and comprises from 2 to 5, from 5 to 10,or from 10 to 15 amino acid substitutions or is otherwise identical tothe amino acid sequence of SEQ ID NO:2 or the to the correspondingparental AAV capsid protein sequence. In other embodiments, the variantAAV capsid comprises (i) a peptide insertion comprising the amino acidsequence QADTTKN (SEQ ID NO:23) or comprising, consisting essentiallyof, or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO:39)between amino acids 587 and 588 of VP1 of AAV2 or the correspondingamino acids of another AAV capsid and (ii) a G453R substitution relativeto the amino acid sequence of AAV2 capsid (SEQ ID NO:2) or thecorresponding substitution in another AAV parental serotype andcomprises from 2 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions or is otherwise identical to the amino acid sequence ofSEQ ID NO:2 or to the corresponding parental AAV capsid proteinsequence. In another embodiment, the variant capsid comprises a peptideinsertion comprising the amino acid sequence QADTTKN (SEQ ID NO:23) orcomprising, consisting essentially of, or consisting of the amino acidsequence LAQADTTKNA (SEQ ID NO:39) between amino acids 587 and 588 ofAAV2 capsid and is otherwise identical to the amino acid sequence setforth in SEQ ID NO:2. In some embodiments, the variant AAV capsid has anamino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98% sequence identity to or is 100%identical to the following amino acid sequence:

(SEQ ID NO: 58) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTEEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAQADTTKNA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In some embodiments, the variant AAV capsid comprises (i) a peptideinsertion comprising the amino acid sequence TNRTSPD (SEQ ID NO:24) orcomprising, consisting essentially of, or consisting of the amino acidsequence LATNRTSPDA (SEQ ID NO:40) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid and(ii) a V708I amino acid substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding the correspondingsubstitution in another AAV parental serotype and comprises from 2 to 5,from 5 to 10, or from 10 to 15 amino acid substitutions or is otherwiseidentical to the amino acid sequence of SEQ ID NO:2 or the to thecorresponding parental AAV capsid protein sequence. In some embodiments,the variant AAV capsid has an amino acid sequence having at least about85%, at least about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 59) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTEEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LATNRTSPDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNY NKS INVDFTVDTNGVYSEPRPIGTRYLTRNL

In some embodiments, the variant AAV capsid comprises a peptideinsertion comprising the amino acid sequence SNTTQKT (SEQ ID NO:25) orcomprising, consisting essentially of, or consisting of the amino acidsequence LASNTTQKTA (SEQ ID NO:41) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid andis otherwise identical to the amino acid sequence of SEQ ID NO:2 or theto the corresponding parental AAV capsid protein sequence. In someembodiments, the variant AAV capsid has an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about98% sequence identity to or is 100% identical to the following aminoacid sequence:

(SEQ ID NO: 60) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LASNTTQKTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In some embodiments, the variant AAV capsid comprises (i) a peptideinsertion comprising the amino acid sequence ASDSTKA (SEQ ID NO:26) orcomprising, consisting essentially of, or consisting of the amino acidsequence LAASDSTKAA (SEQ ID NO:42) between amino acids 587 and 588 ofVP1 of AAV2 or the corresponding amino acids of another AAV capsid and(ii) a V708I amino acid substitution relative to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) the corresponding substitution in anotherAAV parental serotype, wherein the substituted amino acid(s) do notnaturally occur at the corresponding positions, and comprises from 2 to5, from 5 to 10, or from 10 to 15 amino acid substitutions or isotherwise identical to the amino acid sequence of SEQ ID NO:2 or to thecorresponding parental AAV capsid protein sequence. In anotherembodiment, the variant capsid comprises a peptide insertion comprisingthe amino acid sequence ASDSTKA (SEQ ID NO:26) or comprising, consistingessentially of, or consisting of the amino acid sequence LAASDSTKAA (SEQID NO:42) between amino acids 587 and 588 of AAV2 capsid and isotherwise identical to the amino acid sequence set forth in SEQ ID NO:2.In some embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 61) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTEEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAASDSTKAA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL.

In several aspects, a variant AAV capsid protein is provided comprisingone or more amino acid substitutions relative to a correspondingparental AAV capsid protein, wherein the variant capsid protein, whenpresent in an AAV virion, confers increased infectivity of a muscle cell(e.g. a skeletal or cardiac muscle cell) compared to the infectivity ofa muscle cell by an AAV virion comprising the corresponding parental AAVcapsid protein.

In some embodiments a variant AAV capsid protein comprises an amino acidsubstitution at amino acid 363 compared to the amino acid sequence ofAAV2 capsid (SEQ ID NO:2) or the corresponding position in another AAVparental serotype (i.e. other than AAV2). In some preferred embodiments,the variant capsid protein comprises an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about98%, or at least about 99%, or greater, amino acid sequence identity tothe entire length of the amino acid sequence set forth in SEQ ID NO 2and comprises an amino acid substitution at amino acid 363 compared tothe amino acid sequence of AAV2 capsid (SEQ ID NO:2). In some preferredembodiments, a variant AAV capsid protein comprises a P363L amino acidsubstitution compared to the amino acid sequence of AAV2 capsid (SEQ IDNO:2), AAV3A capsid (SEQ ID NO:3) or AAV3B capsid (SEQ ID NO:4); or aP364L amino acid substitution compared to the amino acid sequence ofAAV1 capsid (SEQ ID NO:1) or AAV6 capsid (SEQ ID NO: 7); or a P354Lamino acid substitution compared to the amino acid sequence of AAV4capsid (SEQ ID NO:5) or AAV5 capsid (SEQ ID NO:6); or a P365L amino acidsubstitution compared to the amino acid sequence of AAV7 capsid (SEQ IDNO:8) or AAV9 capsid (SEQ ID NO:10); or a P366L amino acid substitutioncompared to the amino acid sequence of AAV8 capsid (SEQ ID NO:9) orAAV10 capsid (SEQ ID NO:11). In some preferred embodiments, the variantcapsid protein comprises a P363L substitution compared to the amino acidsequence of SEQ ID NO:2, or the corresponding substitution compared toany of SEQ ID NOs: 1 and 3-12, and has at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or at least about99%, or greater, amino acid sequence identity to the entire length of anamino acid sequence set forth in SEQ ID NO:2, or any of SEQ ID NOs: 1and 3-12. In some preferred embodiments, the variant capsid proteincomprises an amino acid sequence comprising a P363L amino acidsubstitution compared to the amino acid sequence set forth in SEQ IDNO:2 and is otherwise identical to the amino acid sequence set forth inSEQ ID NO:2. In related embodiments, the variant capsid proteincomprises a P363L amino acid substitution compared to the amino acidsequence of SEQ ID NO:2, or the corresponding substitution in anotherAAV parental serotype (i.e. other than AAV2) wherein the variant capsidprotein comprises from 1 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions compared to the amino acid sequence of an AAV2 capsidprotein set forth in SEQ ID NO:2 or compared to the amino acid sequenceof a capsid protein in another AAV parental serotype. In anotherpreferred embodiment, the variant capsid comprises a P363L amino acidsubstitution and further comprises E347K and/or V708I amino acidsubstitution(s) compared to the amino acid sequence of SEQ ID NO:2 orthe corresponding substitutions in a capsid from another AAV parentalserotype (i.e. other than AAV2). In another preferred embodiment, thevariant capsid comprises a P363L amino acid substitution compared to theamino acid sequence of SEQ ID NO:2 or the corresponding substitution ina capsid from another AAV parent serotype and further comprises apeptide insertion, preferably located between amino acids 587 and 588 ofVP1 of AAV2, amino acids 588 and 589 of AAV3A, AAV3B, AAV9, or AAV10,amino acids 589 and 590 of VP1 of AAV7, amino acids 590 to 591 of VP1 ofAAV1, AAV6, or AAV8, amino acids 584 and 585 of VP1 of AAV4, or aminoacids 575 and 576 of AAV5, wherein the peptide insertion preferablycomprises an amino acid sequence selected from NKIQRTD (SEQ ID NO:13),NKTTNKD (SEQ ID NO:14), TNKIGVT (SEQ ID NO:15), GNLTKGN (SEQ ID NO:16),NTVKLST (SEQ ID NO:17), SNTVKAI (SEQ ID NO:18), ASNITKA (SEQ ID NO:19),DNTVTRS (SEQ ID NO:20), NKISAKD (SEQ ID NO:21), NQDYTKT (SEQ ID NO:22),QADTTKN (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), SNTTQKT (SEQ ID NO:25),ASDSTKA (SEQ ID NO:26), LANKIQRTDA (SEQ ID NO:27), LANKTTNKDA (SEQ IDNO:28), LATNKIGVTA (SEQ ID NO:29), LAGNLTKGNA (SEQ ID NO:30), LANTVKLSTA(SEQ ID NO:31), LASNTVKAIA (SEQ ID NO:32), LAASNITKAA (SEQ ID NO:33),LADNTVTRSA (SEQ ID NO:34), LANKISAKDA (SEQ ID NO:35), LANQDYTKTA (SEQ IDNO:36), LATNKIGVTS (SEQ ID NO:37), LATNKIGVTA (SEQ ID NO:38), LAQADTTKNA(SEQ ID NO:39), LATNRTSPDA (SEQ ID NO:40), LASNTTQKTA (SEQ ID NO:41),and LAASDSTKAA (SEQ ID NO:42), more preferably selected from GNLTKGN(SEQ ID NO:16), LAGNLTKGNA (SEQ ID NO:30), QADTTKN (SEQ ID NO:23) andLAQADTTKNA (SEQ ID NO:39), and optionally comprises from 2 to 5, from 5to 10, or from 10 to 15 amino acid substitutions or is otherwiseidentical to the amino acid sequence of SEQ ID NO:2 or the to thecorresponding parental AAV capsid protein sequence.

In other embodiments a variant AAV capsid protein comprises an aminoacid substitution at amino acid 593 compared to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding position in anotherAAV parental serotype (i.e. other than AAV2). In some preferredembodiments, the variant capsid protein comprises an amino acidsubstitution at amino acid 593 compared to the amino acid sequence ofAAV2 capsid (SEQ ID NO:2) and has at least about 85%, at least about90%, at least about 95%, at least about 98%, or at least about 99%, orgreater, amino acid sequence identity to the entire length of the aminoacid sequence set forth in SEQ ID NO 2 or is otherwise identical to theamino acid sequence set forth in SEQ ID NO: 2. In some embodiments, thevariant capsid protein comprises a glycine to glutamate amino acidsubstitution at amino acid 594 compared to the amino acid sequence ofAAV1, AAV3A, AAV6, or AAV9, or at amino acid 583 of AAV5, or at aminoacid 596 of AAV8 or AAV10, or an arginine to glutamate amino acidsubstitution at amino acid 594 of AAV3B, or an aspartate to glutamateamino acid substitution at amino acid 592 of AAV4 or a glutamine toglutamate amino acid substitution at position 595 of AAV7. In otherembodiments, the variant capsid protein comprises an A593E amino acidsubstitution compared to the amino acid sequence of AAV2 and does notcomprise one or more of the following amino acid substitutions comparedto the amino acid sequence of AAV2: 119V, V369A, K26R, N215D, G355S,V46A and S196P. In related embodiments, the variant capsid proteincomprises A593E and V708I amino acid substitutions compared to the aminoacid sequence of AAV2 and has at least about 85%, at least about 90%, atleast about 95%, at least about 98% or at least about 99% identity tothe entire length of the amino acid sequence set forth in SEQ ID NO 2 oris otherwise identical to the amino acid sequence set forth in SEQ IDNO:2. In related embodiments, the variant capsid protein comprises A593Eand S109T amino acid substitutions compared to the amino acid sequenceof AAV2 and has at least about 85%, at least about 90%, at least about95%, at least about 98% or at least about 99% identity to the entirelength of the amino acid sequence set forth in SEQ ID NO 2 or isotherwise identical to the amino acid sequence set forth in SEQ ID NO:2.In related embodiments, the variant capsid protein comprises A593E,V708I and S109T amino acid substitutions compared to the amino acidsequence of AAV2 and has at least about 85%, at least about 90%, atleast about 95%, at least about 98% or at least about 99% identity tothe entire length of the amino acid sequence set forth in SEQ ID NO 2 oris otherwise identical to SEQ ID NO:2. In other embodiments, the variantcapsid comprises A593E, V708I and N551S amino acid substitutionscompared to the amino acid sequence of AAV2 and has at least about 85%,at least about 90%, at least about 95%, at least about 98% or at leastabout 99% identity to the entire length of the amino acid sequence setforth in SEQ ID NO 2 or is otherwise identical to the amino acidsequence set forth in SEQ ID NO:2. In other embodiments, the the variantcapsid comprises A593E, V708I and K649E amino acid substitutionscompared to the amino acid sequence of AAV2 and has at least about 85%,at least about 90%, at least about 95%, at least about 98% or at leastabout 99% identity to the entire length of the amino acid sequence setforth in SEQ ID NO 2 or is otherwise identical to the amino acidsequence set forth in SEQ ID NO:2. In other embodiments, the the variantcapsid comprises A593E, V708I, S109T and K527Q amino acid substitutionscompared to the amino acid sequence of AAV2 and has at least about 85%,at least about 90%, at least about 95%, at least about 98% or at leastabout 99% identity to the entire length of the amino acid sequence setforth in SEQ ID NO 2 or is otherwise identical to the amino acidsequence set forth in SEQ ID NO:2.

In other embodiments a variant AAV capsid protein comprises an aminoacid substitution at amino acid 708 compared to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding position in anotherAAV parental serotype (i.e. other than AAV2) wherein the substitutedamino acid does not naturally occur at the corresponding position.Preferably, the rAAV virion does not comprise a proline to serinesubstitution at amino acid 250 compared to AAV2 or a corresponding aminoacid in another AAV parental serotype. In some embodiments, the variantcapsid protein comprises an amino acid substitution at amino acid 708compared to the amino acid sequence of AAV2 capsid (SEQ ID NO:2) and hasat least about 85%, at least about 90%, at least about 95%, at leastabout 98%, or at least about 99%, or greater, amino acid sequenceidentity to the entire length of the amino acid sequence set forth inSEQ ID NO 2 or is otherwise identical to SEQ ID NO:2. In preferredembodiments, the variant capsid protein comprises a valine to isoleucine(V708I) substitution at amino acid 708 compared to the amino acidsequence of AAV2 capsid and has at least about 85%, at least about 90%,at least about 95%, at least about 98%, or at least about 99%, orgreater, amino acid sequence identity to the entire length of the aminoacid sequence set forth in SEQ ID NO 2 or is otherwise identical to theamino acid sequence of SEQ ID NO:2, wherein the variant capsid proteindoes not comprise a P250S amino acid substitution. In some embodiments,the variant capsid protein comprises a valine to isoleucine substitutionat amino acid 709 of AAV3A or AAV3B, an alanine to isoleucinesubstitution at position 709 of AAV1 or AAV6, an asparagine toisoleucine substitution at amino acid 707 of AAV4 or amino acid 709 ofAAV9 or a threonine to isoleucine substitution at amino acid 710 of AAV7or amino acid 711 of AAV8 or AAV10 or a glutamine to isoleucinesubstitution at amino acid 697 of AAV5. In related embodiments, thevariant capsid protein comprises a V708I amino acid substitutioncompared to the amino acid sequence of AAV2, wherein the variant capsidprotein comprises from 2 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions and wherein the variant capsid protein does not comprise aP250S amino acid substitution. In other embodiments, the variant capsidprotein comprises a V708I amino acid substitution and also comprises anA333S and/or S721L amino acid substitution compared to the amino acidsequence of AAV2. In other related embodiments, the variant capsidcomprises a V708I amino acid substitution and also comprises an A333Sand/or S721L amino acid substitution compared to the amino acid sequenceof AAV2 and has at least about 85%, at least about 90%, at least about95%, at least about 98%, or at least about 99%, or greater, amino acidsequence identity to the entire length of the amino acid sequence setforth in SEQ ID NO 2 or is otherwise identical to the amino acidsequence of SEQ ID NO:2.

In other embodiments, a variant AAV capsid protein comprises an aminoacid sequence at least 85%, at least 90%, at least 95% or at least 98%identical to a wild-type AAV capsid sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 10,11 and 12 and alsocomprises i) one or more amino acid substitutions selected from thegroup consisting of A35P, D213N, A319T+P195L, P363L, P363L+V708I, G453R,R651H, I698V, V708I, V708I+A593, V708I+S109T, V708I+T330A, V708I+R588M,V708I+W694C, V708I+W606C, V708I+N449K, V708I+G222S,N312K+N449D+N551S+1698V+L735Q, N312K+N449D+N551S+1698V+V708I+L735Q,and/or (ii) a peptide insertion selected from the group consisting ofNKIQRTD (SEQ ID NO:13), NKTTNKD (SEQ ID NO:14), TNKIGVT (SEQ ID NO:15),GNLTKGN (SEQ ID NO:16), NTVKLST (SEQ ID NO:17), SNTVKAI (SEQ ID NO:18),ASNITKA (SEQ ID NO:19), DNTVTRS (SEQ ID NO:20), NKISAKD (SEQ ID NO:21),NQDYTKT (SEQ ID NO:22), QADTTKN (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24),SNTTQKT (SEQ ID NO:25), ASDSTKA (SEQ ID NO:26), LANKIQRTDA (SEQ IDNO:27), LANKTTNKDA (SEQ ID NO:28), LATNKIGVTA (SEQ ID NO:29), LAGNLTKGNA(SEQ ID NO:30), LANTVKLSTA (SEQ ID NO:31), LASNTVKAIA (SEQ ID NO:32),LAASNITKAA (SEQ ID NO:33), LADNTVTRSA (SEQ ID NO:34), LANKISAKDA (SEQ IDNO:35), LANQDYTKTA (SEQ ID NO:36), LATNKIGVTS (SEQ ID NO:37), LATNKIGVTA(SEQ ID NO:38), LAQADTTKNA (SEQ ID NO:39), LATNRTSPDA (SEQ ID NO:40),LASNTTQKTA (SEQ ID NO:41), and LAASDSTKAA (SEQ ID NO:42). In someembodiments, the variant AAV capsid comprises the specified one or moreamino acid substitutions and/or peptide insertions and is otherwiseidentical to a sequence selected from the group consisting of SEQ IDNOS: 1-12.

In some embodiments, a variant AAV capsid protein is an ancestral capsidprotein comprising one or more peptide insertion(s) and/or amino acidsubstitutions as herein described. By an ancestral capsid protein it ismeant an evolutionary ancestor of a capsid protein that is found innature today, e.g. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAVrh10, AAV11, AAV12, AAV13, which is generated in silico by randomamino acid substitution at positions of degeneracy between AAV capsidproteins that are found in nature today.

In other embodiments, a variant AAV capsid protein is a chimeracomprising amino acids 130-725 of AAV5 capsid (SEQ ID NO:6) or an aminoacid sequence at least 90%, at least 95% or at least 98% identicalthereto.

In some aspects, a variant AAV capsid protein is a chimera comprising(i) amino acids 1-129 of AAV6 (SEQ ID NO:7) or an amino acid sequence atleast 90%, at least 95% or at least 98% identical thereto and (ii) aminoacids 130-725 of AAV5 (SEQ ID NO:6) or an amino acid sequence at least90%, at least 95% or at least 98% identical thereto and furthercomprising V229I, A490T and A581T and optionally V447F or Y585S aminoacid substitutions relative to the sequence of AAV5 (SEQ ID NO:6). Insome embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 62) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDR I VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAF T TTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNR VAYNVGGQMATNNQSSTTAPT TGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTR PIGTRYLTRPL

In other aspects, a variant AAV capsid protein is a chimera comprising(i) amino acids 1-61 of AAV2 (SEQ ID NO:2) or an amino acid sequence atleast 90%, at least 95% or at least 98% identical thereto, (ii) aminoacids 62-129 of AAV6 (SEQ ID NO:7) or an amino acid sequence at least90%, at least 95% or at least 98% identical thereto, and (iii) aminoacids 130-725 of AAV5 (SEQ ID NO:6) and further comprising V229I, A490Tand A581T amino acid substitutions relative to the sequence of AAV5 (SEQID NO:6). In some embodiments, the variant AAV capsid has an amino acidsequence having at least about 85%, at least about 90%, at least about95%, at least about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 63) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDR I VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAF T TTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNR VAYNVGGQMATNNQSSTTAPT TGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTR PIGTRYLTRPL

The AAV variants disclosed herein were generated through the use of invivo directed evolution involving the use of primate cardiac andskeletal muscle screens following intravenous administration. In someembodiments, the variant capsid proteins disclosed herein, when presentin an AAV virion, confer increased transduction of a muscle cellcompared to the transduction of the muscle cell by an AAV virioncomprising the corresponding parental AAV capsid protein or wild-typeAAV. For example, in some embodiments, the variant capsid proteinsdisclosed herein, when present in an AAV virion, confer more efficienttransduction of primate muscle cells than AAV virions comprising thecorresponding parental AAV capsid protein or wild-type AAV capsidprotein, e.g. the muscle cells take up more AAV virions comprising thesubject variant AAV capsid protein than AAV virions comprising theparental AAV capsid protein or wild-type AAV. In some such embodiments,the AAV variant virion or variant rAAV exhibits at least 2-fold, atleast 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, atleast 25-fold, at least 50-fold, or more than 50-fold, increasedtransduction of a muscle cell, compared to the transduction of themuscle cell by a wild-type AAV virion or rAAV comprising thecorresponding parental AAV capsid protein. In preferred embodiments, thethe AAV variant virion or variant rAAV exhibits at least 2-fold, atleast 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, atleast 25-fold, at least 50-fold, at least 100-fold, at least 1000-foldor more than 1000-fold, increased transduction of a muscle cell,compared to the transduction of the muscle cell by a wild-type AAV8 orAAV9 virion. In certain such embodiments, the variant capsid proteinsdisclosed herein, when present in an AAV virion, confer broadertransduction of the primate muscle cells than AAV virions comprising thecorresponding parental AAV capsid protein or wild type AAV capsidprotein. In other words, the variant AAV virion transduces cell typesnot transduced by virions comprising the corresponding parental AAVcapsid protein, and hence more types of cells in the muscle than thecorresponding parental AAV virion. In some embodiments, the AAV variantvirion preferentially transduces a muscle cell, e.g., a subject rAAVvirion infects a muscle cell with 2-fold, 5-fold, 10-fold, 15-fold,20-fold, 25-fold, 50-fold, or more than 50-fold, specificity thananother muscle cell or a non-muscle cell. In some embodiments, thetransduced muscle cell is a cardiac muscle cell (e.g. cardiomyocte,cardiac fibroblast, or a cardiac progenitor cell). In some embodiments,the muscle cell is a skeletal muscle cell (e.g. a myoblast, a myotube ora satellite cell). An increase in transduction of a muscle cell, e.g.increased efficiency of transduction, broader transduction, morepreferential transduction, etc. may be readily assessed in vitro or invivo by any number of methods in the art for measuring gene expression.For example, the AAV may be packaged with a genome comprising anexpression cassette comprising a reporter gene, e.g. a fluorescentprotein, under the control of a ubiquitous or tissue specific promoter,and the extent of transduction assessed by detecting the fluorescentprotein by, e.g., fluorescence microscopy. As another example, the AAVmay be packaged with a genome comprising a barcoded nucleic acidsequence, and the extent of transduction assessed by detecting thenucleic acid sequence by, e.g., PCR. As another example, the AAV may bepackaged with a genome comprising an expression cassette comprising atherapeutic gene for the treatment of a muscle disease, and the extentof transduction assessed by detecting the treatment of the muscledisease in an afflicted patient that was administered the AAV.

Diseases that can be treated using a variant rAAV vector or virionand/or method disclosed herein include, but are not limited to,monogenic diseases, complex diseases, and traumatic injuries. Examplesof monogenic diseases include, but are not limited to, musculardystrophies such as Duchenne, Becker, congenital (including, but notlimited to Bethlem myopathy, Ullrich muscular dystrophy, Fukuyamamuscular dystrophy, Integrin-Deficient, merosin-deficient musculardystrophy, and Walker-Warburgh syndrome), distal (including, but notlimited to Gowers-Laing, Miyoshi, and Nonaka), Emery-Dreifuss,facioscapulohumeral, limb girdle, myotonic and muscular dystrophies;myotonia congenita and paramyotonia congenita; myotubular myopathy;centronuclear myopathy; myofibrillary myopathy, desmin related; anemia;Andersen-Tawil syndrome; Nemaline myopathy; Brody disease; lysosomalstorage disorders such as alpha-mannosidosis, aspartylglucosaminuria,beta-mannosidosis, cystinosis, Farber disease, fucosidosis, Gaucherdisease, galactosialidosis, gangliosidoses (including, but not limitedto AB variant, activator deficiency, beta-galactosidase deficiency,Fabry disease, Sandhoff disease, and Schindler disease), glycogenstorage disorders (including, but not limited to as Andersen disease,Cori disease, Danon disease, Forbes disease, glucose-6-phosphate defect,Hers disease, lactate dehydrogenase A deficiency, Pompe disease, Taruidisease, and von Gierke disease), infantile free sialic acid storagedisease, lysosomal acid lipase deficiency, Krabbe disease, MetachromaticLeukodystrophy, mucopolysaccharidoses (including, but not limited tohyaluronidase deficiency, Hunter syndrome, Hurler syndrome,Hurler-Scheie syndrome, Maroteaux-Lamy syndrome, Morquio syndrome,Sanfilippo syndrome, Scheie syndrome, and Sly syndrome), mucolipidosis(including, but not limited to Sialidosis, I-cell disease, mucolipidin 1deficiency, and Psuedy-Hurler Polydystrophy), multiple sulfasedeficiency, Niemann-Pick disease, neuronal ceroid lipofuscinoses(including, but not limited to Batten-Spielmeyer-Vogt disease,congenital Cathepsin D deficiency, German/Serbian Late Infantile,Jansky-Bielschowsky disease, Kufs disease, late infantile, lateinfantile variant, Northern Epilepsy, Santavuori-Haltia disease, andTurkish Late Infantile), pyknodysostosis, Salla disease, Saposin Bdeficiency, Tay-Sach's disease and Wolman disease; metabolic disorderssuch as adenosine monophosphate deaminase deficiency, alkaptonuria,carnitine deficiency, carnitine palmityl transferase deficiency, Hartnupdisorder, homocystinuria, maple syrup urine disease, myophosphorylasedeficiency, phosphofuctokinase deficiency, phosphoglycerate kinasedeficiency, phosphoglycerate mutase deficiency, phosphorylasedeficiency, and Tangier disease; Friedreich's ataxia; ataxiatalengiectasia; ataxia with vitamin E deficiency; periodic paralysis,such as Gamstorp disease and hypokalemic periodic paralysis;mitochondrial diseases such as Barth syndrome, Kearns-Sayre syndrome,mitochondrial myopathy, mitochondrial encephalopathy lactic acidosis andstroke-like episodes, myoclonic epilepsy with ragged-red fibers, andPearson syndrome; familial hypertrophic cardiomyopathies; dilatedcardiomyopathies; familial congenital heart diseases, such as familialaortic valve disease and non-compaction of the left ventricle withcongenital heart defects; familial arrhythmias, such as Andersoncardiodysrhythmic periodic paralysis, atrial septal defects with AVconduction defects, Brugada syndrome, cardiac conductance defect,catecholaminergic polymorphic ventricular tachycardia, and congenitalheart block; familial vascular disorders, such as arterial tortuositysyndrome, cerebral autosomal dominant arteriopathy with sobcorticalinfacts and leukoenceophalopathy, cerebral recessive dominantarteriopathy with sobcortical infacts and leukoenceophalopathy, familialtype aortic aneurysm, Marfan syndrome, Ehlers-Danlos syndrome, Bealscongenital contractual arachnodactyly, Loeys-Dietz syndrome, andpseudoxanthoma elasticum; arrhythmogenic right ventricularcardiomyopathy; familial arrhythmogenic right ventricular dysplasia;Naxos disease; left ventricular non-compaction; familial atrialfibrillation; familial ventricular tachycardia; familialWolff-Parkinson-White syndrome; long QT syndromes; short QT syndrome;sick sinus syndromes; lipoprotein diseases, such as abetalipoproteinemiaand lipoprotein lipase deficiency; alpha-1 antitrypsin deficiency;coagulation factor VIII deficiency (hemophilia A) or coagulation factorIX deficiency (hemophilia B); thalassemia; fibrodysplasia ossificansprogressive; laminopathies; Huntington disease; congenital myasthenicsyndromes; Hutchinson-Gilford Progeria syndrome; Noonan syndrome;congenital fibre type disproportion myopathy; congenital fibrosis of theextraocular muscles; minicore myopathy; rippling muscle disease;Schwartz-Jampel syndrome; tubular aggregate myopathy; and zebra bodymyopathy Examples of complex diseases include, but are not limited to,heart/cardiovascular disease (e.g. congestive heart failure, myocardialinfarction, angina, coronary artery disease, ischaemic heart disease,cardiomyopathy); cancer; diabetes; and infection. Examples of traumaticinjuries include, but are not limited to, viral infection of the muscle,muscle laceration; and muscle contusion. In preferred embodiments, avariant rAAV vector or virion and/or method disclosed herein is used totreat Fabry disease, Friedreich's ataxia, Duchenne muscular dystrophy,Becker muscular dystrophy, Pompe disease, myophosphorylase deficiency,facioscapulohumeral muscular dystrophy, limb girdle muscular dystrophy,or myotonic dystrophy.

In another embodiment, a variant capsid disclosed herein comprises aheterologous nucleic acid comprising a nucleotide sequence encoding agene product such as, without limitation, an interfering RNA, a longnon-coding RNA, a short non-coding RNA, an antisense RNA, an aptamer, apolypeptide, a secreted antibody, a single chain antibody, a VHH domain,a soluble receptor, an affibody, a knottin, a DARPin, a centurin, achaperone, a site-specific nuclease that provides for site-specificknock-down of gene function or a modified site-specific nuclease thatprovides for gene-specific activation of transcription.

A rAAV variant virion disclosed herein comprises a heterologous nucleicacid comprising a nucleotide sequence encoding a gene product. In someembodiments, the gene product is an antisense RNA, a microRNA (miRNA), ashort hairpin RNA (shRNA) or a small interfering RNA (siRNA) or aprecursor or mimic thereof. In some embodiments, the gene product is along non-coding RNA. In some embodiments, the gene product is a shortnon-coding RNA. In some embodiments, the gene product is an antisenseRNA. In some embodiments, the gene product is an aptamer. In someembodiments, the gene product is a polypeptide. In some embodiments, thegene product is a secreted antibody. In some embodiments, the geneproduct is a single chain antibody. In some embodiments, the geneproduct is a VHH domain. In some embodiments, the gene product is asoluble receptor. In some embodiments, the gene product is an affibody.In some embodiments, the gene product is a knottin. In some embodiments,the gene product is a DARPin. In some embodiments, the gene product is acenturin. In some embodiments, the gene product is a chaperone. In someembodiments, the gene product is a site-specific nuclease that providefor site-specific knock-down of gene function.

The uses of the gene product include, but are not limited to, enhancingthe level of a factor in a cell, enhancing the level of a factor in aneighboring or distant cell through secretion of a factor, decreasingthe level of a factor in a cell, or decreasing the level of a factor ina neighboring or distant cell through secretion of a factor. The geneproduct can be designed to supplement the level of a defective ofmissing gene product, decrease the level of a defective of missing geneproduct, introduce a new supporting gene product, supplement the levelof a supporting gene product, decrease the level of a hindering geneproduct, or both decrease the level of a hindering gene product andintroduce or supplement the level of a supporting gene product.

Gene products delivered by the subject AAV variants can be used to alterthe level of gene products or gene product activity directly orindirectly linked to muscle diseases and trauma. Skeletal, cardiac orsmooth muscle transduced with subject AAV variants can also be used as abiofactory to produce and secrete therapeutic proteins for the treatmentof diseases in trans in distant organs. Genes whose gene products aredirectly or indirectly linked to genetic diseases include, e.g., genesencoding any of the following gene products: dystrophin including mini-and micro-dystrophins (DMD; e.g. GenBank Accession Number NP_003997.1;SEQ ID NO:64); titin (TTN); titin cap (TCAP) α-sarcoglycan (SGCA),β-sarcoglycan (SGCB), γ-sarcoglycan (SGCG) or δ-sarcoglycan (SGCD);alpha-1-antitrypsin (A1-AT); myosin heavy chain 6 (MYH6); myosin heavychain 7 (MYH7); myosin heavy chain 11 (MYH11); myosin light chain 2(ML2); myosin light chain 3 (ML3); myosin light chain kinase 2 (MYLK2);myosin binding protein C (MYBPC3); desmin (DES); dynamin 2 (DNM2);laminin α2 (LAMA2); lamin A/C (LMNA); lamin B (LMNB); lamin B receptor(LBR); dysferlin (DYSF); emerin (EMD); insulin; blood clotting factors,including but not limited to, factor VIII and factor IX; erythropoietin(EPO); lipoprotein lipase (LPL); sarcoplasmic reticulum Ca2⁺⁺-ATPase(SERCA2A), 5100 calcium binding protein A1 (S100A1); myotubularin (MTM);DM1 protein kinase (DMPK; e.g. GenBank Accession Number NG 009784.1; SEQID NO:65); glycogen phosphorylase L (PYGL); glycogen phosphorylase,muscle associated (PYGM; e.g. GenBank Accession Number NP_005600.1; SEQID NO:66); glycogen synthase 1 (GYS1); glycogen synthase 2 (GYS2);α-galactosidase A (GLA; e.g. GenBank Accession Number NP_000160.1; SEQID NO:67); α-N-acetylgalactosaminidase (NAGA); acid α-glucosidase (GAA;e.g. GenBank Accession Number NP_000143.2; SEQ ID NO:68),sphingomyelinase phosphodiesterase 1 (SMPD1); lysosomal acid lipase(LIPA); collagen type I α1 chain (COL1A1); collagen type I α2 chain(COL1A2); collagen type III α1 chain (COL3A1); collagen type V α1 chain(COL5A1); collagen type V α2 chain (COL5A2); collagen type VI α1 chain(COL6A1); collagen type VI α2 chain (COL6A2); collagen type VI α3 chain(COL6A3); procollagen-lysine 2-oxoglutarate 5-dioxygenase (PLOD1);lysosomal acid lipase (LIPA); frataxin (FXN; e.g. GenBank AccessionNumber NP_000135.2; SEQ ID NO:69); myostatin (MSTN); β-N-acetylhexosaminidase A (HEXA); β-N-acetylhexosaminidase B (HEXB);β-glucocerebrosidase (GBA); adenosine monophosphate deaminase 1 (AMPD1);β-globin (HBB); iduronidase (IDUA); iduronate 2-sulfate (IDS); troponin1 (TNNI3); troponin T2 (TNNT2); troponin C (TNNC1); tropomyosin 1(TPM1); tropomyosin 3 (TPM3); N-acetyl-α-glucosaminidase (NAGLU);N-sulfoglucosamine sulfohydrolase (SGSH); heparan-α-glucosaminideN-acetyltransferase (HGSNAT); integrin α 7 (IGTA7); integrin α 9(IGTA9); glucosamine(N-acetyl)-6-sulfatase (GNS);galactosamine(N-acetyl)-6-sulfatase (GALNS); β-galactosidase (GLB1);β-glucuronidase (GUSB); hyaluronoglucosaminidase 1 (HYAL1); acidceramidase (ASAH1); galactosylcermidase (GALC); cathepsin A (CTSA);cathepsin D (CTSA); cathepsin K (CTSK); GM2 ganglioside activator(GM2A); arylsulfatase A (ARSA); arylsulfatase B (ARSB);formylglycine-generating enzyme (SUMF1); neuraminidase 1 (NEU1);N-acetylglucosamine-1-phosphate transferase α (GNPTA);N-acetylglucosamine-1-phosphate transferase β (GNPTB);N-acetylglucosamine-1-phosphate transferase γ (GNPTG); mucolipin-1(MCOLN1); NPC intracellular transporter 1 (NPC1); NPC intracellulartransporter 2 (NPC2); ceroid lipofuscinosis 5 (CLN5); ceroidlipofuscinosis 6 (CLN6); ceroid lipofuscinosis 8 (CLN8); palmitoylprotein thioesterase 1 (PPT1); tripeptidyl peptidase 1 (TPP1); battenin(CLN3); DNAJ heat shock protein family 40 member C5 (DNAJC5); majorfacilitator superfamily domain containing 8 (MFSD8); mannosidase α class2B member 1 (MAN2B1); mannosidase β (MANBA); aspartylglucosaminidase(AGA); α-L-fucosidase (FUCA1); cystinosin, lysosomal cysteinetransporter (CTNS); sialin; solute carrier family 2 member 10 (SLC2A10);solute carrier family 17 member 5 (SLC17A5); solute carrier family 6member 19 (SLC6A19); solute carrier family 22 member 5 (SLC22A5); solutecarrier family 37 member 4 (SLC37A4); lysosomal associated membraneprotein 2 (LAMP2); sodium voltage-gated channel a subunit 4 (SCN4A);sodium voltage-gated channel β subunit 4 (SCN4B); sodium voltage-gatedchannel α subunit 5 (SCNSA); sodium voltage-gated channel a subunit 4(SCN4A); calcium voltage-gated channel subunit α1c (CACNA1C); calciumvoltage-gated channel subunit als (CACNA1S); phosphoglycerate kinase 1(PGK1); phosphoglycerate mutase 2 (PGAM2);amylo-α-1,6-glucosidase,4-α-glucanotransferase (AGL); potassiumvoltage-gated channel ISK-related subfamily member 1 (KCNE1); potassiumvoltage-gated channel ISK-related subfamily member 2 (KCNE2); potassiumvoltage-gated channel subfamily J member 2 (KCNJ2); potassiumvoltage-gated channel subfamily J member 5 (KCNJ5); potassiumvoltage-gated channel subfamily H member 2 (KCNH2); potassiumvoltage-gated channel KQT-like subfamily member 1 (KCNQ1);hyperpolarization-activated cyclic nucleotide-gated potassium channel 4(HCN4); chloride voltage-gated channel 1 (CLCN1); carnitinepalmitoyltransferase 1A (CPT1A); ryanodine receptor 1 (RYR1); ryanodinereceptor 2 (RYR2); bridging integrator 1 (BIN1); LARGE xylosyl- andglucuronyltransferase 1 (LARGE1); docking protein 7 (DOK7); fukutin(FKTN); fukutin related protein (FKRP); selenoprotein N (SELENON);protein O-mannosyltransferase 1 (POMT1); protein O-mannosyltransferase 2(POMT2); protein O-linked mannose N-acetylglucosaminyltransferase 1(POMGNT1); protein O-linked mannose N-acetylglucosaminyltransferase 2(POMGNT2); protein-O-mannose kinase (POMK); isoprenoid synthase domaincontaining (ISPD); plectin (PLEC); cholinergic receptor nicotinicepsilon subunit (CHRNE); choline 0-acetyltransferase (CHAT); cholinekinase β (CHKB); collagen like tail subunit of asymmetricacetylcholinesterase (COLQ); receptor associated protein of the synapse(RAPSN); four and a half LIM domains 1 (FHL1);β-1,4-glucuronyltransferase 1 (B4GAT1);β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2); dystroglycan 1(DAG1); transmembrane protein 5 (TMEM5); transmembrane protein 43(TMEM43); SECIS binding protein 2 (SECISBP2); glucosamine(UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE); anoctamin 5(ANO5); structural maintenance of chromosomes flexible hinge domaincontaining 1 (SMCHD1); lactate dehydrogenase A (LDHA); lactatedehydrogenase B (LHDB); calpain 3 (CAPN3); caveolin 3 (CAV3); tripartitemotif containing 32 (TRIM32); CCHC-type zinc finger nucleic acid bindingprotein (CNBP); nebulin (NEB); actin, α1, skeletal muscle (ACTA1);actin, α1, cardiac muscle (ACTC1); actinin α2 (ACTN2); poly(A)-bindingprotein nuclear 1 (PABPN1); LEM domain-containing protein 3 (LEMD3);zinc metalloproteinase STE24 (ZMPSTE24); microsomal triglyceridetransfer protein (MTTP); cholinergic receptor nicotinic α1 subunit(CHRNA1); cholinergic receptor nicotinic α2 subunit (CHRNA2);cholinergic receptor nicotinic α3 subunit (CHRNA3); cholinergic receptornicotinic α4 subunit (CHRNA4); cholinergic receptor nicotinic α5 subunit(CHRNA5); cholinergic receptor nicotinic α6 subunit (CHRNA6);cholinergic receptor nicotinic α7 subunit (CHRNA7); cholinergic receptornicotinic α8 subunit (CHRNA8); cholinergic receptor nicotinic α9 subunit(CHRNA9); cholinergic receptor nicotinic α10 subunit (CHRNA10);cholinergic receptor nicotinic β1 subunit (CHRNB1); cholinergic receptornicotinic β2 subunit (CHRNB2); cholinergic receptor nicotinic β3 subunit(CHRNB3); cholinergic receptor nicotinic β4 subunit (CHRNB4);cholinergic receptor nicotinic γ subunit (CHRNG1); cholinergic receptornicotinic ∂ subunit (CHRND); cholinergic receptor nicotinic ε subunit(CHRNE1); ATP binding cassette subfamily A member 1 (ABCA1); ATP bindingcassette subfamily C member 6 (ABCC6); ATP binding cassette subfamily Cmember 9 (ABCC9); ATP binding cassette subfamily D member 1 (ABCD1);ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1 (ATP2A1);ATM serine/threonine kinase (ATM); a tocopherol transferase protein(TTPA); kinesin family member 21A (KIF21A); paired-like homeobox 2a(PHOX2A); heparan sulfate proteoglycan 2 (HSPG2); stromal interactionmolecule 1 (STIM1); notch 1 (NOTCH1); notch 3 (NOTCH3); dystrobrevin α(DTNA); protein kinase AMP-activated, noncatalytic γ2 (PRKAG2);cysteine- and glycine-rich protein 3 (CSRP3); viniculin (VCL); myozenin2 (MyoZ2); myopalladin (MYPN); junctophilin 2 (JPH2); phospholamban(PLN); calreticulin 3 (CALR3); nexilin F-actin-binding protein (NEXN);LIM domain binding 3 (LDB3); eyes absent 4 (EYA4); huntingtin (HTT);androgen receptor (AR); protein tyrosine phosphate non-receptor type 11(PTPN11); junction plakoglobin (JUP); desmoplakin (DSP); plakophilin 2(PKP2); desmoglein 2 (DSG2); desmocollin 2 (DSC2); catenin α3 (CTNNA3);NK2 homeobox 5 (NKX2-5); A-kinase anchor protein 9 (AKAP9); A-kinaseanchor protein 10 (AKAP10); guanine nucleotide-binding proteinα-inhibiting activity polypeptide 2 (GNAI2); ankyrin 2 (ANK2);syntrophin α-1 (SNTA1); calmodulin 1 (CALM1); calmodulin 2 (CALM2); HTRAserine peptidase 1 (HTRA1); fibrillin 1 (FBN1); fibrillin 2 (FBN2);xylosyltransferase 1 (XYLT1); xylosyltransferase 2 (XYLT2); tafazzin(TAZ); homogentisate 1,2-dioxygenase (HGD); glucose-6-phosphatasecatalytic subunit (G6PC); 1,4-alpha-glucan enzyme 1 (GBE1);phosphofructokinase, muscle (PFKM); phosphorylase kinase regulatorysubunit alpha 1 (PHKA1); phosphorylase kinase regulatory subunit alpha 2(PHKA2); phosphorylase kinase regulatory subunit beta (PHKB);phosphorylase kinase catalytic subunit gamma 2 (PHKG2); phosphoglyceratemutase 2 (PGAM2); cystathionine-beta-synthase (CBS);methylenetetrahydrofolate reductase (MTHFR);5-methyltetrahydrofolate-homocysteine methyltransferase (MTR);5-methyltetrahydrofolate-homocysteine methyltransferase reductase(MTRR); methylmalonic aciduria and homocystinuria, cblD type (MMADHC);mitochondrial DNA, including, but not limited to mitochondrially encodedNADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1); mitochondriallyencoded NADH:ubiquinone oxidoreductase core subunit 5 (MT-ND5);mitochondrially encoded tRNA glutamic acid (MT-TE); mitochondriallyencoded tRNA histadine (MT-TH); mitochondrially encoded tRNA leucine 1(MT-TLi); mitochondrially encoded tRNA lysine (MT-TK); mitochondriallyencoded tRNA serine 1 (MT-TS1); mitochondrially encoded tRNA valine(MT-TV); mitogen-activated protein kinase kinase 1 (MAP2K1); B-Rafproto-oncogene, serine/threonine kinase (BRAF); raf-1 proto-oncogene,serine/threonine kinase (RAF1); growth factors, including, but notlimited to insulin growth factor 1 (IGF-1); transforming growth factor03 (TGFβ3); transforming growth factor β receptor, type I (TGFβR1);transforming growth factor β receptor, type II (TGFβR2), fibroblastgrowth factor 2 (FGF2), fibroblast growth factor 4 (FGF4), vascularendothelial growth factor A (VEGF-A), vascular endothelial growth factorB (VEGF-B); vascular endothelial growth factor C (VEGF-C), vascularendothelial growth factor D (VEGF-D), vascular endothelial growth factorreceptor 1 (VEGFR1), and vascular endothelial growth factor receptor 2(VEGFR2); interleukins; immunoadhesins; cytokines; and antibodies.

In preferred embodiments, gene products delivered by the subject AAVvariants are selected from alpha galactosidase A (GLA), Frataxin (FXN),Dystrophin (DMD), Acid alpha glucosidase (GAA), and Glycogenphosphorylase, muscle (PYGM). In some preferred embodiments, a subjectAAV variant comprises a nucleic acid segment comprising a nucleotidesequence encoding (i) a GLA polypeptide comprising or consisting of theamino acid sequence set forth as SEQ ID NO:67, (ii) an FXN polypeptidecomprising or consisting of the amino acid sequence set forth as SEQ IDNO:69, (iii) a DMD polypeptide comprising or consisting of a functionalfragment (e.g. mini or micro dystrophin, preferably comprising an intactactin-binding domain, at least 4 of the 24 spectrin-like repeats and thedystroglycan-binding domain) of the amino acid sequence set forth as SEQID NO:64, (iv) a GAA polypeptide comprising or consisting of the aminoacid sequence set forth as SEQ ID NO:68, (v) a PYGM polypeptidecomprising or consisting of the amino acid sequence set forth as SEQ IDNO:66, (vi) or (v) an amino acid sequence at least 80%, at least 85%, atleast 90% or at least 95% identical to any one of SEQ ID NOs:64 and66-69.

In another preferred embodiment, a subject AAV variant comprises atransgene encoding an interfering RNA, e.g. an antisense RNA, an miRNA,an shRNA, or an siRNA, that decreases the expression of DMPK. In someaspects, the interfering RNA decreases the expression of DMPK encoded bya nucleic acid having a nucleotide sequence as set forth as SEQ ID NO:65or a sequence at least 80%, at least 85%, at least 90%, or at least 95%identical to SEQ ID NO:65.

Genes whose gene products induce or promote apoptosis are referred toherein as “pro-apoptotic genes” and the products of those genes (mRNA;protein) are referred to as “pro-apoptotic gene products.” Pro-apoptotictargets include, e.g., Bax gene products; Bid gene products; Bak geneproducts; Bad gene products; Bcl-2; Bcl-X1. Anti-apoptotic gene productsinclude X-linked inhibitor of apoptosis.

Genes whose gene products induce or promote angiogenesis are referred toherein as “pro-angiogenic genes” and the products of those genes (mRNA;protein) are referred to as “pro-angiogenic gene products.”Pro-angiogenic targets include, e.g., vascular endothelial growth factor(VEGFa, VEGFb, VEGFc, VEGFd); vascular endothelial growth factorreceptor 1 (VEGFR1); vascular endothelial growth factor receptor 2(VEGFR2); Fms-Related Tyrosine Kinase 1 (Flt1); placenta growth factor(PGF); Platelet-derived growth factor (PDGF); angiopoietins; sonichedgehog. Genes whose gene products inhibit angiogenesis are referred toherein as “anti-angiogenic genes” and the products of those genes (mRNA;protein) are referred to as “anti-angiogenic gene products.”Anti-angiogenic gene products include endostatin; tumstatin;angiostatin; pigment epithelium-derived factor (PEDF), and fusionproteins or antibodies that are specific for pro-angiogenic targetsand/or their receptors, e.g. the VEGF-specific antibody Avastin™, etc.

Genes whose gene products function as immune modulators, e.g.,complement factors, toll-like receptors, are called “immunomodulatorygenes”. Exemplary immunomodulatory genes include cytokines, chemokines,and the fusion proteins or antibodies that are specific for them and/ortheir receptors, e.g. the anti-IL-6 fusion protein Rilonaceptn™, theComplement Factor H-specific antibody lampamizumab, etc. Genes whosegene products function as muscle protective factors, e.g., insulingrowth factor 1 (IGF-1); transforming growth factor β (TGFβ); fibroblastgrowth factor (FGF).

In some cases, a gene product of interest is a site-specificendonuclease that provide for site-specific knock-down of gene function,e.g., where the endonuclease knocks out an allele associated with amuscle disease. For example, where a dominant allele encodes a defectivecopy of a gene that, when wild-type, is a muscle structural proteinand/or provides for normal muscle function, a site-specific endonucleasecan be targeted to the defective allele and knock out the defectiveallele.

In addition to knocking out a defective allele, a site-specific nucleasecan also be used to stimulate homologous recombination with a donor DNAthat encodes a functional copy of the protein encoded by the defectiveallele. Thus, e.g., a subject rAAV virion can be used to deliver both asite-specific endonuclease that knocks out a defective allele, and canbe used to deliver a functional copy of the defective allele, resultingin repair of the defective allele, thereby providing for production of afunctional muscle protein (e.g., functional lamin A/C, functionalfibrillin, functional collagen type VI, etc.). In some embodiments, arAAV virion disclosed herein comprises a heterologous nucleotidesequence that encodes a site-specific endonuclease; and a heterologousnucleotide sequence that encodes a functional copy of a defectiveallele, where the functional copy encodes a functional muscle protein.Functional muscle proteins include, e.g., lamin A/C, fibrillin 1,COL6A1, COL6A2, COL6A3, and the like.

Site-specific endonucleases that are suitable for use include, e.g.,meganucleases; zinc finger nucleases (ZFNs); transcriptionactivator-like effector nucleases (TALENs); and Clustered regularlyinterspaced short palindromic repeats/CRISPR-associated (Cas), wheresuch site-specific endonucleases are non-naturally occurring and aremodified to target a specific gene. Such site-specific nucleases can beengineered to cut specific locations within a genome, and non-homologousend joining can then repair the break while inserting or deletingseveral nucleotides. Such site-specific endonucleases (also referred toas “INDELs”) then throw the protein out of frame and effectively knockout the gene. See, e.g., U.S. Patent Publication No. 2011/0301073.

In some embodiments of the variant rAAV vector disclosed herein, anucleotide sequence encoding a gene product of interest is operablylinked to a constitutive promoter. Suitable constitutive promotersinclude e.g. cytomegalovirus promoter (CMV) (Stinski et al. (1985)Journal of Virology 55(2): 431-441), CMV early enhancer/chicken β-actin(CBA) promoter/rabbit β-globin intron (CAG) (Miyazaki et al. (1989) Gene79(2): 269-277, CB^(SB) (Jacobson et al. (2006) Molecular Therapy 13(6):1074-1084), human elongation factor 1α promoter (EF1α) (Kim et al.(1990) Gene 91(2): 217-223), human phosphoglycerate kinase promoter(PGK) (Singer-Sam et al. (1984) Gene 32(3): 409-417, mitochondrialheavy-strand promoter (Loderio et al. (2012) PNAS 109(17): 6513-6518),ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101-105). Inother embodiments, a nucleotide sequence encoding a gene product ofinterest is operably linked to an inducible promoter. In some instances,a nucleotide sequence encoding a gene product of interest is operablylinked to a tissue-specific or cell type-specific regulatory element.For example, in some instances, a nucleotide sequence encoding a geneproduct of interest is operably linked to a muscle-specific regulatoryelement (e.g., a cardiac specific promoter or a skeletal muscle specificpromoter), e.g., a regulatory element that confers selective expressionof the operably linked gene in a muscle cell. Suitable muscle-specificregulatory elements include, e.g., skeletal muscle α-actin promoter(Muscat and Kedes (1987) Mol. Cell. Biol. 7:4089-4099); cardiac muscleα-actin promoter (Minty and Kedes (1986) Mol. Cell. Biol. 6:2125-2136);smooth muscle α-actin promoter (Nakano et al. (1991) Gene 99:285-289);vascular smooth muscle α-actin promoter (Keogh et al. (1999) GeneTherapy 6(4):616-628); muscle creatine kinase promoter (Bartlett et al.(1996) Cell Transplantation 5(3):411-419); myosin light chain 1 andmyosin light chain 3 promoters (Seidel and Arnold (1989) J. Biol. Chem.264(27):16109-16117); myosin light chain 2v (MLC2v) promoter (Su et al.(2004) PNAS 101(46):16280-16285); myogenic factor 5 (Myf5) promoter(Fujimaki et al. (2004) Journal of Biological Chemistry289(11):7399-7412); myogenic differentiation 1 (Myod1) promoter (Zingget al. (1994) Nucleic Acids Research 22(12):2234-2241); myogenin (Myog)promoter (Salminen et al. (1991). Journal of Cell Biology115(4):905-917); paired box gene 7 (Pax7) promoter (Murmann et al.(2000) Biol Chem. 381(4):331-335); paired like homeodomain 3 (Pitx3)promoter (Coulon et al. (2007) Journal of Biological Chemistry282:33192-33200); MHCK7 promoter (Salva et al. (2007) Mol. Ther.15(2):320-329); MCK/SV40 promoter (Takeshita et al. (2007) InternationalJournal of Molecular Medicine 19:309-315); C5-12 promoter (Li et al.(1999) Nature Biotechnology 17:241-245); double and triple tandem MCKenhancer/promoters (Wang et al. (2008) Gene Therapy 15:1489-1499);myosin heavy chain 7 (MYH7) promoter; (Iwaki et al. (2104) PLoS ONE9(4):e88610); myosin heavy chain 6 (MYH6) promoter (Pacak et al. (2008)Genet. Vaccines Ther. 6:13); cardiac troponin T(TNNT2) promoter (Farzaet al. (1998)J. Mol. Cell Cardiol. 30(6):1247-53); α-tropomyosinpromoter (Helfman et al. (1986) Molecular and Cellular Biology6(11):3582-3595); cardiac troponin C (TNNC1) promoter (Scheier et al.(1990) Journal of Biological Chemistry 34(5):21247-21253); cardiacmyosin-binding protein C promoter (Lin et al. (2013) PLoS ONE 8(7):e69671); cardiac troponin I (TNNI3) promoter (Bhavsar et al. (1996)Genomics 35(1):11-23); the desmin promoter (Li et al. (1991) Journal ofBiological Chemistry 10(5):6562-6570); sodium-calcium exchanger (NCX1)promoter (Scheller et al. (1997) Journal of Biological Chemistry273(13):7643-7649); atrial natriuretic factor promoter (Durocher et al.(1996) Molecular and Cellular Biology 16(9):4648-4655); and SM22apromoter (Kemp et al. (1995) Biochemical Journal 310(3):1037-1043.

For the purposes of the invention, the disclosure herein provides anisolated nucleic acid comprising a nucleotide sequence that encodes avariant AAV capsid protein as described above. An isolated nucleic acidcan be an AAV vector, e.g., a recombinant AAV vector.

The disclosure herein also provides a method of treating a muscledisease, the method comprising administering to an individual in needthereof an effective amount of a rAAV variant virion comprising atransgene of interest as described above and disclosed herein. One ofordinary skill in the art would be readily able to determine aneffective amount of the subject rAAV virion and that the disease hadbeen treated by testing for a change in one or more functional oranatomical parameters, e.g. muscle biopsy followed byimmunohistochemistry, serum sampling followed by ELISA or enzymeactivity assays, walk test, peak maximum oxygen consumption, biomarkeranalysis left ventricular ejection fraction, left ventricularend-systolic volume, hand-held dynamometry, maximum weight lift, TimedFunction Tests, the Hammersmith Motor Ability Score, timed rise fromfloor, or 9 Hole Peg Test.

Nonlimiting methods for assessing muscle function and changes thereofinclude assessing walk test, peak maximum oxygen consumption, biomarkeranalysis, left ventricular ejection fraction, left ventricularend-systolic volume, Vignos Scale, Timed Function Tests, the HammersmithMotor Ability Score, timed rise from floor, Motor Function MeasureScale, North Star Ambulatory Assessment, 9 Hole Peg Test, or Children'sHospital of Philadelphia Infant Test of Neuromuscular Disorders.

In some embodiments, an effective amount of the subject rAAV virionresults in a decrease in the rate of loss of muscle function, anatomicalmuscle integrity, or muscle mass, e.g. a 2-fold, 3-fold, 4-fold, or5-fold or more decrease in the rate of loss and hence progression ofdisease, for example, a 10-fold decrease or more in the rate of loss andhence progression of disease. In some embodiments, the effective amountof the subject rAAV virion results in a gain in muscle function, gain inmuscle strength, gain in muscle mass, and/or an improvement inanatomical muscle integrity or biomarkers, e.g. a 2-fold, 3-fold, 4-foldor 5-fold improvement or more in muscle function, muscle strength,muscle mass, and/or improvement in anatomical muscle integrity orbiomarkers, e.g. a 10-fold improvement or more in muscle function,muscle strength, muscle mass and/or improvement in anatomical muscleintegrity or biomarkers. As will be readily appreciated by theordinarily skilled artisan, the dose required to achieve the desiredtreatment effect will typically be in the range of 1×10⁸ to about 1×10¹⁶recombinant virions, typically referred to by the ordinarily skilledartisan as 1×10⁸ to about 1×10¹⁶ “vector genomes” and preferably will bein the range of about 1×10¹¹ to about 1×10¹⁵ recombinant virions.

A subject rAAV virion can be delivered to skeletal muscle byintravascular (intravenous or intra-arterial) administration,intraperitoenal administration, limb perfusion and/or directintramuscular injection or by any other convenient mode or route ofadministration that will result in the delivery of the rAAV virion toskeletal muscle. The rAAV virion can be delivered to cardiac muscle byintravascular (intravenous or intra-arterial) administration, directcardiac injection (into the left atrium, right atrium, right ventricleand/or septum), antegrade or retrograde infusion into the coronaryartery (via the left anterior descending or left circumflex coronaryarteries), recirculation, intrapericardial injection, transendocardialinjection, or by any other convenient mode or route of administrationthat will result in the delivery of the rAAV virion to cardiac muscle.In a preferred embodiment, a subject rAAV virion is delivered toskeletal and/or cardiac muscle by systemic intravenous administration.When administered via intravenous injection, the subject rAAV virion isable to move through the circulatory system and transduce muscle cellsmore efficiently, compared to the capability of a wild type AAV virionor an AAV virion comprising the corresponding parental AAV capsidprotein. #

A variant capsid protein disclosed herein is isolated, e.g., purified.In some embodiments, a variant capsid protein disclosed herein isincluded in an AAV vector or a recombinant AAV (rAAV) virion. In otherembodiments, such AAV variant vectors and/or AAV variant virions areused in an in vivo or ex vivo method of treating a muscle disease inprimate cardiac or skeletal muscle.

The disclosure herein further provides host cells such as, withoutlimitation, isolated (genetically modified) host cells comprising asubject nucleic acid. A host cell according to the invention disclosedherein, can be an isolated cell, such as a cell from an in vitro cellculture. Such a host cell is useful for producing a subject rAAV variantvirion, as described herein. In one embodiment, such a host cell isstably genetically modified with a nucleic acid. In other embodiments, ahost cell is transiently genetically modified with a nucleic acid. Sucha nucleic acid is introduced stably or transiently into a host cell,using established techniques, including, but not limited to,electroporation, calcium phosphate precipitation, liposome-mediatedtransfection, and the like. For stable transformation, a nucleic acidwill generally further include a selectable marker, e.g., any of severalwell-known selectable markers such as neomycin resistance, and the like.Such a host cell is generated by introducing a nucleic acid into any ofa variety of cells, e.g., mammalian cells, including, e.g., murinecells, and primate cells (e.g., human cells). Exemplary mammalian cellsinclude, but are not limited to, primary cells and cell lines, whereexemplary cell lines include, but are not limited to, HEK293 cells,HEK293T cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts,C3H10T1/2 fibroblasts, CHO cells, and the like. Exemplary host cellsinclude, without limitation, HeLa cells (e.g., American Type CultureCollection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61,CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells(e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No.CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No.CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonickidney (HEK293) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. Ahost cell can also be made using a baculovirus to infect insect cellssuch as Sf9 cells, which produce AAV (see, e.g., U.S. Pat. No.7,271,002; U.S. patent application Ser. No. 12/297,958). In someembodiments, a genetically modified host cell includes, in addition to anucleic acid comprising a nucleotide sequence encoding a variant AAVcapsid protein, as described above, a nucleic acid that comprises anucleotide sequence encoding one or more AAV rep proteins. In otherembodiments, a host cell further comprises an rAAV variant vector. AnrAAV variant virion can be generated using such host cells. Methods ofgenerating an rAAV virion are described in, e.g., U.S. PatentPublication No. 2005/0053922 and U.S. Patent Publication No.2009/0202490.

The disclosure herein additionally provides a pharmaceutical compositioncomprising: a) the rAAV variant virion, as described above and disclosedherein; and b) a pharmaceutically acceptable carrier, diluent,excipient, or buffer. In some embodiments, the pharmaceuticallyacceptable carrier, diluent, excipient, or buffer is suitable for use ina human or non-human patient. Such excipients, carriers, diluents, andbuffers include any pharmaceutical agent that can be administeredwithout undue toxicity. Pharmaceutically acceptable excipients include,but are not limited to, liquids such as water, saline, glycerol andethanol. Pharmaceutically acceptable salts can be included therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like.Additionally, auxiliary substances, such as wetting or emulsifyingagents, surfactants, pH buffering substances, and the like, may bepresent in such vehicles. A wide variety of pharmaceutically acceptableexcipients are known in the art and need not be discussed in detailherein. Pharmaceutically acceptable excipients have been amply describedin a variety of publications, including, for example, A. Gennaro (2000)“Remington: The Science and Practice of Pharmacy,” 20th edition,Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and DrugDelivery Systems (1999) H. C. Ansel et al., eds., 7^(th) ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd)ed. Amer.Pharmaceutical Assoc. In some aspects of the present invention, thepresent invention provides a pharmaceutical composition comprising about1×10⁸ to about 1×10¹⁶ recombinant viruses or 1×10⁸ to about 1×10¹⁶vector genomes, wherein each said recombinant virus comprises a genomeencoding one or more gene products.

Some embodiments of the invention are exemplified in the following items1 to 54:

1. A variant adeno-associated virus (AAV) capsid protein comprising apeptide insertion in the GH-loop of the capsid protein, wherein theinsertion is in AAV2 or a corresponding position in a capsid portion ofa wild-type AAV serotype other than AAV2 or an AAV variant, and whereinthe peptide insertion is selected from the group consisting of NKIQRTD(SEQ ID NO:13), NKTTNKD (SEQ ID NO:14), TNKIGVT (SEQ ID NO:15), GNLTKGN(SEQ ID NO:16), NTVKLST (SEQ ID NO:17), SNTVKAI (SEQ ID NO:18), ASNITKA(SEQ ID NO:19), DNTVTRS (SEQ ID NO:20), NKISAKD (SEQ ID NO:21), NQDYTKT(SEQ ID NO:22), QADTTKN (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), SNTTQKT(SEQ ID NO:25), ASDSTKA (SEQ ID NO:26), LANKIQRTDA (SEQ ID NO:27),LANKTTNKDA (SEQ ID NO:28), LATNKIGVTA (SEQ ID NO:29), LAGNLTKGNA (SEQ IDNO:30), LANTVKLSTA (SEQ ID NO:31), LASNTVKAIA (SEQ ID NO:32), LAASNITKAA(SEQ ID NO:33), LADNTVTRSA (SEQ ID NO:34), LANKISAKDA (SEQ ID NO:35),LANQDYTKTA (SEQ ID NO:36), LATNKIGVTS (SEQ ID NO:37), LATNKIGVTA (SEQ IDNO:38), LAQADTTKNA (SEQ ID NO:39), LATNRTSPDA (SEQ ID NO:40), LASNTTQKTA(SEQ ID NO:41), and LAASDSTKAA (SEQ ID NO:42).

2. The variant AAV of item 1, wherein the capsid protein comprises oneor more point mutations relative to AAV2 or one or more correspondingpoint mutations relative to other wild-type AAV serotypes or AAVvariants.

3. The variant AAV of item 2, wherein the one or more point mutations isselected from the group consisting of A35P, S109T, P195L, D213N, G222S,V229L, N312K, A319T, T330A, A333S, E347K, P363L, A427D, V447F, N449D,N449K?, G453R, A490T, K527Q, N551S, A581T, Y585S, R588M, A593E, W606C,K649E, R651H, W694C, 1698V, V708I, and L735Q, and is preferably selectedfrom the group consisting of V708L, V708I+A593E, V708I+S109T,V708I+T330A, A35P, V708I+R588M, V708I+W606C, V708I+W694C, 1698V,N312K+N449D+N551S+I698V+L735Q, N312K+N449D+N551S+I698V+V708I+L735Q,V708I+N449K, and V708I+G222S.

4. The variant AAV of item 1, wherein the peptide insertion is insertedfollowing any of amino acids in positions 570-671 in VP1 of AAV2 or acorresponding position in another wild-type AAV serotype or AAV variant.

5. The variant AAV of item 4, wherein the peptide insertion is insertedfollowing amino acid 587 in VP1 of AAV2 or a corresponding position inanother AAV serotype.

6. An infectious recombinant adeno-associated virus (rAAV) virioncomprising: (a) a variant AAV capsid protein according to any one ofitems 1-5, and a heterologous nucleic acid.

7. The rAAV of item 6, wherein the heterologous nucleic acid comprises anucleotide sequence encoding an RNA interfering agent or a polypeptide.

8. A method of delivering a heterologous nucleic acid to a target cells,comprising contacting a target cell with the rAAV virion of item 7.

9. The method of item 8, wherein the target cell is a cardiac and/orskeletal muscle cell.

10. The method of item 8, wherein the target cell is in vitro.

11. The method of item 8, wherein the target cell is in vivo.

12. An isolated nucleic acid comprising a nucleotide sequence encoding avariant adeno-associated virus (AAV) capsid protein comprising a peptideinsertion in the GH-loop of the capsid protein, wherein the insertion isin AAV2 or a corresponding position in a capsid portion of a wild-typeAAV serotype other than AAV2 or an AAV variant, and wherein the peptideinsertion is selected from the group consisting of NKIQRTD (SEQ IDNO:13), NKTTNKD (SEQ ID NO:14), TNKIGVT (SEQ ID NO:15), GNLTKGN (SEQ IDNO:16), NTVKLST (SEQ ID NO:17), SNTVKAI (SEQ ID NO:18), ASNITKA (SEQ IDNO:19), DNTVTRS (SEQ ID NO:20), NKISAKD (SEQ ID NO:21), NQDYTKT (SEQ IDNO:22), QADTTKN (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), SNTTQKT (SEQ IDNO:25), ASDSTKA (SEQ ID NO:26), LANKIQRTDA (SEQ ID NO:27), LANKTTNKDA(SEQ ID NO:28), LATNKIGVTA (SEQ ID NO:29), LAGNLTKGNA (SEQ ID NO:30),LANTVKLSTA (SEQ ID NO:31), LASNTVKAIA (SEQ ID NO:32), LAASNITKAA (SEQ IDNO:33), LADNTVTRSA (SEQ ID NO:34), LANKISAKDA (SEQ ID NO:35), LANQDYTKTA(SEQ ID NO:36), LATNKIGVTS (SEQ ID NO:37), LATNKIGVTA (SEQ ID NO:38),LAQADTTKNA (SEQ ID NO:39), LATNRTSPDA (SEQ ID NO:40), LASNTTQKTA (SEQ IDNO:41), and LAASDSTKAA (SEQ ID NO:42).

13. An isolated host cell comprising the nucleic acid of item 12.

14. A variant adeno-associated virus (AAV) capsid protein comprising apeptide insertion relative to a parental AAV capsid proteincorresponding to two adjacent amino acids at a position between aminoacids 570 and 611 of VP1 of AAV2, wherein the insertion comprises theamino acid sequence Y₁Y₂X₁X₂X₃X₄X₅X₆X₇Y₃, and wherein X₁ is selectedfrom T and N; X₂ is selected from N and K; X₃ is selected from K, I andT; X₄ is selected from I, Q and T; X₅ is selected from G, R and N; X₆ isselected from V, T and K; and X₇ is selected from T and D.

15. The variant AAV of item 14, wherein the peptide insertion isselected from the group consisting of NKIQRTD (SEQ ID NO:13), NKTTNKD(SEQ ID NO:14) and TNKIGVT (SEQ ID NO:15).

16. The variant AAV of item 15, wherein the peptide insertion is flankedby N-terminus amino acids LA and C-terminus amino acid A.

17. The variant AAV of item 15, wherein the peptide insertion is betweenamino acids 587 and 588 of VP1 of AAV2 or a corresponding position inanother wild-type AAV serotype or AAV variant.

18. An infectious recombinant adeno-associated virus (rAAV) virioncomprising: (a) a variant AAV capsid protein according to any one ofitems 14-17, and a heterologous nucleic acid.

19. The rAAV of item 18, wherein the heterologous nucleic acid comprisesan RNA interfering agent or a nucleotide sequence encoding apolypeptide.

20. A method of delivering a heterologous nucleic acid to a target cell,comprising contacting the target cell with the rAAV virion of item 18.

21. The method of item 20, wherein the target cell is a cardiac and/orskeletal cell.

22. The method of item 21, wherein the target cell is in vitro or invivo.

23. A variant adeno-associated virus (AAV) capsid protein comprising i)an AAV amino acid sequence at least 90% identical to a wild-type AAVselected from the group consisting of SEQ ID NOS: 1-10 and 11; and ii)one or more amino acid substitutions selected from the group consistingof P363L, P363L+V708I, P363L+E347K, V708I+A593E, V708I+A333S,V708I+S721L, V708I+A593E+N551S, V708I+A593E+K649E, V708I+A593E+S109T,V708I+A593E+S109T+K527Q, A593E+S109T, wherein the one or moresubstitutions are relative to AAV2 or the one or more correspondingsubstitutions relative to other AAV serotypes.

24. The variant AAV of item 23, wherein the capsid protein comprises apeptide insertion.

25. The variant AAV of item 24, wherein the peptide insertion isselected from the group consisting of NKIQRTD (SEQ ID NO:13), NKTTNKD(SEQ ID NO:14), TNKIGVT (SEQ ID NO:15), GNLTKGN (SEQ ID NO:16), NTVKLST(SEQ ID NO:17), SNTVKAI (SEQ ID NO:18), ASNITKA (SEQ ID NO:19), DNTVTRS(SEQ ID NO:20), NKISAKD (SEQ ID NO:21), NQDYTKT (SEQ ID NO:22), QADTTKN(SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), SNTTQKT (SEQ ID NO:25), ASDSTKA(SEQ ID NO:26), LANKIQRTDA (SEQ ID NO:27), LANKTTNKDA (SEQ ID NO:28),LATNKIGVTA (SEQ ID NO:29), LAGNLTKGNA (SEQ ID NO:30), LANTVKLSTA (SEQ IDNO:31), LASNTVKAIA (SEQ ID NO:32), LAASNITKAA (SEQ ID NO:33), LADNTVTRSA(SEQ ID NO:34), LANKISAKDA (SEQ ID NO:35), LANQDYTKTA (SEQ ID NO:36),LATNKIGVTS (SEQ ID NO:37), LATNKIGVTA (SEQ ID NO:38), LAQADTTKNA (SEQ IDNO:39), LATNRTSPDA (SEQ ID NO:40), LASNTTQKTA (SEQ ID NO:41), andLAASDSTKAA (SEQ ID NO:42).

26. The variant AAV of item 23, wherein the AAV amino acid sequence isat least 95% identical to the wild-type AAV.

27. The variant AAV of item 23, wherein the AAV amino acid sequence isat least 99% identical to the wild-type AAV.

28. The variant AAV of item 23, wherein the capsid protein is a chimericcapsid protein or is an ancestral capsid protein.

29. An infectious recombinant adeno-associated virus (rAAV) virioncomprising: (a) a variant AAV capsid protein according to any one ofitems 23-28, and a heterologous nucleic acid.

30. The rAAV of item 29, wherein the heterologous nucleic acid comprisesa nucleotide sequence encoding an RNA interfering agent or apolypeptide.

31. A method of delivering a heterologous nucleic acid to a target cell,comprising contacting the target cell with the rAAV virion of item 29.

32. The method of item 31, wherein the target cell is a cardiac and/orskeletal muscle cell.

33. The method of item 32, wherein the cardiac cell is selected from thegroup consisting of cardiomyocytes, cardiomyoblasts, cardiacfibroblasts, and cardiac progenitor cells.

34. The method of item 31, wherein the target cell is in vitro.

35. The method of item 31, wherein the target cell is in vivo.

36. An isolated nucleic acid comprising a nucleotide sequence encoding avariant adeno-associated virus (AAV) capsid protein comprising an aminoacid sequence at least 90% identical to a wild-type AAV selected fromthe group consisting of SEQ ID NO:1-12 or an AAV variant; and ii) one ormore amino acid substitutions selected from the group consisting ofP363L, P363L+V708I, P363L+E347K, V708I+A593E, V708I+A333S, V708I+S721L,V708I+A593E+N551S, V708I+A593E+K649E, V708I+A593E+S109T,V708I+A593E+S109T+K527Q, A593E+S109T.

37. An isolated host cell comprising the nucleic acid of item 36.

38. A variant adeno-associated virus (AAV) capsid protein comprising apeptide insertion in the GH-loop of the capsid protein and optionallycomprising one or more point mutations, wherein the peptide insertion isselected from the group consisting of NKIQRTD (SEQ ID NO:13) andLANKIQRTDA (SEQ ID NO:26).

39. The variant AAV capsid protein according to item 38, comprising aV708I amino acid substitution.

40. The variant AAV capsid protein according to item 39, comprising aV708I+A593E, V708I+S109T, V708I+T330A, V708I+R588M orV708I+N312K+N449D+N551S+I698V+L735Q amino acid substitution.

41. The variant AAV capsid protein according to item 38, comprising anA35P amino acid substitution.

42. The variant AAV capsid protein according to item 38, comprising aN312K+N449D+N551S+I698V+L735Q amino acid substitution.

43. A variant adeno-associated virus (AAV) capsid protein comprising apeptide insertion in the GH-loop of the capsid protein and optionallycomprising one or more point mutations, wherein the peptide insertion isselected from the group consisting of NKTTNKD (SEQ ID NO:14) andLANKTTNKDA (SEQ ID NO:27).

44. The variant AAV capsid protein according to item 43, comprising aV708I amino acid substitution.

45. The variant AAV capsid protein according to item 44, comprising aV708I+S109T, V708I+W694C, V708I+W606C, orV708I+N312K+N449D+N551S+I698V+L735Q amino acid substitution.

46. The variant AAV capsid protein according to item 43, comprising anI698V amino acid substitution.

47. The variant AAV capsid protein according to item 46, comprising aN312K+N449D+N551S+I698V+L735Q amino acid substitution.

48. A variant adeno-associated virus (AAV) capsid protein comprising apeptide insertion in the GH-loop of the capsid protein and optionallycomprising one or more point mutations, wherein the peptide insertion isselected from the group consisting of TNKIGVT (SEQ ID NO:15), LATNKIGVTA(SEQ ID NO:28) and LATNKIGVTS (SEQ ID NO:36).

49. The variant AAV capsid protein according to item 48, comprising aV708I amino acid substitution.

50. The variant AAV capsid protein according to item 49, comprising aV708I+N449K, V708I+G222S, or V708I+N312K+N449D+N551S+I698V+L735Q aminoacid substitution.

51. The variant AAV capsid protein according to item 48, comprising aN312K+N449D+N551S+I698V+L735Q amino acid substitution.

52. A variant adeno-associated virus (AAV) capsid protein comprising thesequence of SEQ ID NO:62 or a sequence at least 90% identical thereto,wherein the variant AAV capsid proteins comprises the following aminoacid substitutions relative to AAV5 capsid: V229I+A490T+A581T.

53. The variant AAV capsid protein according to item 52, furthercomprising a Y585S or V447F amino acid substitution relative to AA5capsid.

54. A variant adeno-associated virus (AAV) capsid protein comprising thesequence of SEQ ID NO:63 or a sequence at least 90% identical thereto,wherein the variant AAV capsid proteins comprises the following aminoacid substitutions relative to AAV5 capsid: V229I+A427D+A490T+A581T.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference. Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and ClonTech.

Example 1

Intravenous Injection and Tissue Harvesting. A single male cynomolgusmacaque (Macaca fascicularis) age 3-10 years old and weighing at least 3kg was dosed via intravenous injection via the saphenous vein for eachround of selection. The animal was anesthetized and 1-5 mL of thelibrary (in the first round, the library consists of variants generatedusing all mutagenesis techniques described in FIG. 1A; in eachsubsequent round, the variants isolated from the previous round), insome cases pre-incubated with human IVIG for 30 minutes at 37° C., wasadministered.

Euthanasia was performed by trained veterinary staff using 100 mg/kgpentobarbital sodium intravenous injection on day 14±3 or 21±3,depending on the selection. The cardiac and/or skeletal muscle tissuefrom the quadriceps was removed, and DNA was isolated from the tissue.In some cases, the cardiac tissue was divided into several regions: theatrium, ventricular septum, left papillary muscle, right papillarymuscle, left ventricle, and right ventricle.

Directed Evolution. The directed evolution process is shown in FIG.1A-1E. Briefly, a viral capsid library comprising 20+ proprietarycombinations of DNA mutation technique and cap genes is created (FIG.1A). Viruses are then packaged (FIG. 1B)—such that each particle iscomposed of a mutant capsid surrounding the cap gene encoding thatcapsid—and purified. The capsid library is placed under selectivepressure in vivo. The tissue or cellular material of interest isharvested to isolate AAV variants that have successfully infected thattarget, and the successful viruses are recovered. Successful clones areenriched through repeated selection (Stage I—FIG. 1D). Selected capgenes then undergo proprietary re-diversification and are enrichedthrough further selection steps to iteratively increase viral fitness(Stage 2—FIG. 1D). Variants identified during Vector Selection Stages 1and 2 demonstrate the ability to transduce primate muscle cells (FIG.1E).

Successful Recovery of AAV Capsid Genomes. The capsids recovered fromeach round of selection were used to package the library injected toinitiate the subsequent round of selection. Recovery of capsid genesfrom tissue represents successful internalization of library vectorsinto the tissue of interest. Recovery of viral genomes from cardiac andskeletal muscle tissue from a representative round of selection areshown in FIG. 2. Bands within boxes represent successful recovery ofviral genomes.

Sequencing Analysis. During rounds 3-4 of selections including theselective pressure of intravenous delivery to cardiac tissue or skeletalmuscle tissue and rounds 1-2 of a selection including the selectivepressure of intravenous delivery in the presence of neutralizingantibodies to cardiac tissue, sequencing was performed on individualclones within the library to determine the frequency of variants withinthe population. Variants were evaluated for the presence of motifswithin the sequencing data. Variants were grouped into motifs based onthe presence of a unifying variation (for example, a specific pointmutation or specific peptide insertion sequence in a consistent locationwithin the capsid) that occurred in multiple sequences. Motifsrepresenting at least 5% of the sequenced population in two or morerounds of the selection or at least 10% of the sequenced population inone or more rounds of the selection are represented in FIG. 3A (Round 4sequencing analysis for the selective pressure of intravenous deliveryto cardiac tissue), FIG. 3B (Round 2 sequencing analysis for theselective pressure of intravenous delivery in the presence ofneutralizing antibodies to cardiac tissue), and FIG. 3C (provides Round3 sequencing analysis for the selective pressure of intravenous deliveryto skeletal muscle tissue.

Several representative clones that were identified as conferringincreased infectivity of cardiac and/or skeletal muscle cells are listedin Table 1 below (each clone contains the identified substitution(s)and/or peptide insertion and is otherwise identical to SEQ ID NO:2; theselection round, number of sequences and frequency (in parentheses) arelisted for each clone):

TABLE 1 Amino acid sequence modifications to the AAV VP1 capsid proteinthat confer increased infectivity of cardiac and/or skeletalmuscle cells. Substitutions listed in column 2 are based onthe amino acid sequence for wild type AAV2, i.e. in the absenceof inserted peptide. “Cardiac + NAb” in column 5 indicates thatthe amino acid sequence modifications should confer increasedresistance to neutralization by anti-AAV antibodies in addition to increased infectivity of cardiac muscle cells. Cardiac +Skeletal Insertion Substitution Cardiac NAb Muscle 588~LANKIQRTDA~ NoneRound 3: 6 Round 1: 2 Round 3: 1 (SEQ ID NO: 27) (9.68%) (1.41%) (1.23%)Round 4: 11 Round 2: 1 (26.83%) (0.81%) 588~LANKIQRTDA~ +A35P Round 4: 1— — (SEQ ID NO: 27) (2.44%) 588~LANKIQRTDA~ +S109T + V708I Round 3: 1 —— (SEQ ID NO: 27) (1.61%) Round 4: 2 (4.88%) 588~LANKIQRTDA~+R588M + V708I — Round 1: 1 — (SEQ ID NO: 27) (0.70%) 588~LANKIQRTDA~+A593E + V708I Round 3: 1 — — (SEQ ID NO: 27) (1.61%) 588~LANKIQRTDA~+V708I Round 3: 13 Round 1: 1 — (SEQ ID NO: 27) (20.97%) (0.70%)Round 4: 10 (23.26%) 588~LANKTTNKDA~ None Round 4: 2 Round 1: 10 —(SEQ ID NO: 28) (4.88%) (7.04%) Round 2: 8 (6.50%) 588~LANKTTNKDA~+S109T + V708I Round 4: 1 — — (SEQ ID NO: 28) (2.44%) 588~LANKTTNKDA~+W694C + V708I Round 4: 1 — — (SEQ ID NO: 28) (2.44%) 588~LANKTTNKDA~+1698V — Round 1: 1 — (SEQ ID NO: 28) (0.70%) 588~LANKTTNKDA~+W606C + V708I — Round 2: 1 — (SEQ ID NO: 28) (0.81%) 588~LANKTTNKDA~+V708I Round 3: 6 Round 1: 4 — (SEQ ID NO: 28) (9.68%) (2.82%)Round 4: 3 Round 2: 10 (7.32%) (8.13%) 588~LATNKIGYTA~ +V708I Round 4: 1— — (SEQ ID NO: 29) (2.44%) 588~LAQADTTKNA~ None — Round 1: 23 —(SEQ ID NO: 39) (16.02%) Round 2: 21 (17.07%) 588~LAQADTTKNA~ +D213N —Round 2: 1 — (SEQ ID NO: 39) (0.81%) 588~LAQADTTKNA~ +G453R — Round 1: 1— (SEQ ID NO: 39) (0.70%) 588~LAQADTTKNA~ +V708I Round 4: 1 Round 1: 3 —(SEQ ID NO: 39) (2.44%) (2.11%) Round 2: 3 (2.44%) 588~LAQADTTKNA~+P363L — Round 1: 1 — (SEQ ID NO: 39) (0.70%) 588~LANQDYTKTA~ None —Round 1: 1 — (SEQ ID NO: 36) (0.70%) 588~LANQDYTKTA~ +I698V — Round 2: 2— (SEQ ID NO: 36) (1.63%) 588~LANQDYTKTA~ +V708I — Round 1: 1 —(SEQ ID NO: 36) (0.70%) 588~LATNRTSPDA~ +V708I — Round 2: 1 —(SEQ ID NO: 40) (0.81%) 588~LAASDSTKAA~ None — — Round 3: 1(SEQ ID NO: 42) (1.23%) 588~LAASDSTKAA~ +V708I Round 3: 2 — —(SEQ ID NO: 42) (3.23%) 588~LAASNITKAA~ None — Round 1: 2 —(SEQ ID NO: 33) (1.41%) Round 2: 8 (6.50%) 588~LAASNITKAA~ +V708I —Round 1: 6 — (SEQ ID NO: 33) (4.23%) Round 2: 11 (8.94%) 588~LAGNLTKGNA~None Round 3: 4 Round 1: 6 — (SEQ ID NO: 30) (6.44%) (4.23%) Round 2: 3(2.44%) 588~LAGNLTKGNA~ +S109T + V708I Round 3: 2 — — (SEQ ID NO: 30)(3.23%) 588~LAGNLTKGNA~ +A139T + P195L — Round 1: 1 — (SEQ ID NO: 30)(0.70%) 588~LAGNLTKGNA~ +P363L + V708I — Round 1: 1 — (SEQ ID NO: 30)(0.70%) 588~LAGNLTKGNA~ +R651H — Round 2: 1 — (SEQ ID NO: 30) (0.81%)588~LAGNLTKGNA~ +V708I Round 3: 2 Round 1: 1 — (SEQ ID NO: 30) (3.23%)(0.70%) Round 2: 2 (1.63%) 588~LAGNLTKGNA~ +P363L — Round 1: 1 —(SEQ ID NO: 30) (0.70%) 588~LADNTYTRSA~ None — Round 1: 9 —(SEQ ID NO: 34) (6.34%) Round 2: 6 (4.88%) 588~LADNTYTRSA~ +1698V —Round 2: 1 — (SEQ ID NO: 34) (0.81%) 588~LADNTYTRSA~ +V708I — Round 1: 1— (SEQ ID NO: 34) (0.70%) Round 2: 2 (1.63%) 588~LANTYKLSTA~ None —Round 1: 3 — (SEQ ID NO: 31) (2.11%) Round 2: 7 (5.69%) 588~LANTYKLSTA~+V708I — Round 2: 8 — (SEQ ID NO: 31) (6.50%) 588~LASNTYKAIA~ NoneRound 3: 2 — — (SEQ ID NO: 32) (3.23%) Round 4: 1 (2.44%)588~LASNTYKAIA~ +V708I Round 4: 1 — — (SEQ ID NO: 32) (2.44%)588~LATNKIGYTS~ None Round 4: 1 — — (SEQ ID NO: 37) (2.44%)588~LASNTTQKTA~ None — — Round 3: 2 (SEQ ID NO: 41) (2.46%)588~LANKISAKDA~ None — Round 2: 3 — (SEQ ID NO: 35) (2.44%)588~LANKISAKDA~ +V708I — Round 2: 2 — (SEQ ID NO: 35) (1.63%) None P34A— — Round 4: 2 (10%) None P34S — Round 1: 1 — (0.70%) None P64S — —Round 4: 1 (5.00%) None S109T + P235S — — Round 4: 1 (5.00%) None Q120R— — Round 4: 1 (5.00%) None A193V — — Round 3: 1 (1.23%) None T277N — —Round 4: 1 (5.00%) None P351L — — Round 4: 1 (5.00%) None P363L —Round 1: 13 Round 3: 34 (9.15%) (41.98%) Round 4: 4 (20.00%) NoneP363L + E347K — — Round 3: 1 (1.23%) None P363L + V708I — Round 1: 2 —(1.41%) None S427T + I698V — Round 1: 1 — (0.70%) None Q440K — —Round 3: 1 (1.23%) None Y444F — — Round 4: 1 (5.00%) None N449D — —Round 4: 1 (5.00%) None T568N — — Round 3: 1 (1.23%) None A593ERound 4: 3 Round 1: 1 Round 3: 31 (7.32%) (0.70%) (38.27%) Round 4: 2(10%) None S109T + A593E — — Round 3: 2 (2.47%) None S109T + K527Q+Round 3: 1 — — A593E + V708I (1.61%) None S109T + A593E+ Round 3: 1 — —V708I (1.61%) None A593E + N551S+ — — Round 3: 1 V708I (1.23%) NoneA593E + K649E+ Round 3: 1 — — V708I (1.61%) None A593E + V708IRound 3: 12 Round 1: 5 Round 3: 1 (19.35%) (3.52%) (1.23%) None I698V —— Round 4: 1 (5.00%) None V708I Round 3: 6 Round 1: 10 Round 3: 1(9.68%) (7.04%) (1.23%) Round 4: 2 Round 4: 1 (4.88%) (5.00%) NoneV708I + A333S — — Round 3: 1 (1.23%) None V708I + S721L Round 3: 1 — —(1.61%) None V708I + L735V — — Round 3: 1 (1.23%)

Also identified as capsids conferring increased infectivity of cardiacmuscle cell and increased resistance to neutralization by anti-AAVantibodies were the following chimeras:

A chimera with (i) amino acids 1-129 of AAV6 and (ii) amino acids130-725 of AAV5 and having the following amino acid substitutionsrelative to AAV5: V229I+A490T+A581T (the sequence of SEQ ID NO:62).

A chimera with (i) amino acids 1-61 of AAV2, (ii) amino acids 62-129 ofAAV6, and (iii) amino acids 130-725 of AAV5 and having the followingamino acid substitutions relative to AAV5: V229I+A490T+A581T (thesequence of SEQ ID NO:63).

A chimera with (i) amino acids 1-129 of AAV6 and (ii) amino acids130-725 of AAV5 and having the following amino acid substitutionsrelative to AAV5: V229I+A490T+A581T+Y585S

A chimera with (i) amino acids 1-129 of AAV6 and (ii) amino acids130-725 of AAV5 and having the following amino acid substitutionsrelative to AAV5: V229I+A447F+A490T+A581T

The AAV variant virions disclosed herein may incorporate reasonablerational design parameters, features, modifications, advantages, andvariations that are readily apparent to those skilled in the art in thefield of engineering AAV viral vectors.

Example 2

The cell tropism of recombinant AAV virions comprising the novel AAVvariants LANKIQRTDA+V708I (SEQ ID NO:43), LANKTTNKDA+V708I (SEQ IDNO:48), and LATNKIGVTA+V708I (SEQ ID NO:46) for cardiomyocytes wasassessed in vitro using cardiomyocytes generated from human embryonicstem cells (ESC).

Recombinant AAV virions comprising either an AAV1 capsid, an AAV2capsid, an AAV9 capsid, the novel variant capsid LANKIQRTDA+V708I (SEQID NO:43), the novel variant capsid LANKTTNKDA+V708I (SEQ ID NO:48), orthe novel variant capsid LATNKIGVTA+V708I (SEQ ID NO:46) and a genomecomprising a green fluorescent protein (EGFP) transgene operably linkedto a CAG promoter (AAV1.CAG.EGFP, AAV2.CAG.EGFP, AAV9.CAG.EGFP,LANKIQRTDA+V708I (SEQ ID NO:43).CAG.EGFP, LANKTTNKDA+V708I (SEQ IDNO:48).CAG.EGFP, and LATNKIGVTA+V708I (SEQ ID NO:46).CAG.GFP,respectively) were manufactured using standard methods. Cardiomyocyteswere generated from a human embryonic stem cell line, ESI-017, bymodulation of Wnt signaling using small molecules. After 14 days ofcardiac mesoderm induction, cultures were further enriched forcardiomyocytes by glucose deprivation. After approximately 24 days ofdifferentiation, the majority of cells expressed the cardiac myocytemarker, cardiac Troponin T (cTnT), and a ventricular-specific marker,MLC-2V. The generated cardiomyocytes were evaluated for expression ofgap junction protein Connexin 43, membrane potential fluctuation,calcium handling, and contractile function to ensure that the generatedcardiomyocytes reached a mature state prior to vector characterization.

Relative to AAV1, AAV2, and AAV9, the LANKIQRTDA+V708I (SEQ ID NO:43),LANKTTNKDA+V708I (SEQ ID NO:48), and LATNKIGVTA+V708I (SEQ ID NO:46)variants provided for significantly higher transduction efficiency ofand transgene expression in human cardiomyocyte cultures six dayspost-infection as determined by immunofluorescence (FIG. 6A), flowcytometry (FIG. 6B) and Western blot analysis (FIGS. 6C-D). Furthermore,relative to AAV1, AAV2, and AAV9, LANKIQRTDA+V708I (SEQ ID NO:43),LANKTTNKDA+V708I (SEQ ID NO:48), and LATNKIGVTA+V708I (SEQ ID NO:46)provided for faster onset of gene expression in human cardiomyocytecultures, as determined by immunofluorescence (FIG. 6E). Relative toAAV8 and AAV9, which exhibit cardiac and skeletal muscle cell tropism,the number of infectious units per administered viral genome weremultiple orders of magnitude higher for LANKIQRTDA+V708I (SEQ ID NO:43)and LANKTTNKDA+V708I (SEQ ID NO:48) (FIG. 10A). This study illustratesthe superior ability of NKIQRTD (SEQ ID NO:13)-, NKTTNKD (SEQ IDNO:14)-, and TNKIGVT (SEQ ID NO:15)-comprising AAV capsid variants todeliver genes to cardiac cells.

Example 3

The cell tropism of recombinant AAV virions comprising the novel AAVvariant AAV6/AAV5 chimera for cardiomyocytes was assessed in vitro usingcardiomyocytes generated from human embryonic stem cells (ESC).

Recombinant AAV virions comprising either an AAV1 capsid, an AAV8capsid, an AAV9 capsid, or the novel variant capsid AAV6/AAV5 chimera(of SEQ ID NO:62) and a genome comprising a green fluorescent protein(EGFP) transgene operably linked to a CAG promoter (AAV1.CAG.EGFP,AAV8.CAG.EGFP, AAV9.CAG.EGFP, AAV6/AAV5 chimera.CAG.EGFP, respectively)were manufactured using standard methods. Cardiomyocytes were generatedfrom a human embryonic stem cell line, ESI-017, by modulation of Wntsignaling using small molecules. After 14 days of cardiac mesoderminduction, cultures were further enriched for cardiomyocytes by glucosedeprivation. After approximately 24 days of differentiation, themajority of cells expressed the cardiac myocyte marker, cardiac TroponinT (cTnT), and a ventricular-specific marker, MLC-2V. The generatedcardiomyocytes were evaluated for expression of gap junction proteinConnexin 43, membrane potential fluctuation, calcium handling, andcontractile function to ensure that the generated cardiomyocytes reacheda mature state prior to vector characterization.

Relative to AAV1, AAV8, and AAV9, the AAV6/AAV5 chimera provided forsignificantly higher transduction efficiency of and transgene expressionin human cardiomyocyte cultures six days post-infection as determined byimmunofluorescence (FIG. 7A), flow cytometry (FIG. 7B), and Western blotanalysis (FIGS. 7C-D). Furthermore, relative to AAV8, the AAV6/AAV5chimera provided for faster onset of gene expression in humancardiomyocyte cultures, as determined by immunofluorescence (FIG. 7E).Relative to AAV8 and AAV9, the number of infectious units peradministered viral genome were multiple orders of magnitude higher forthe AAV6/AAV5 chimera (FIG. 10A). This study illustrates the superiorability of SEQ ID NO:62-comprising AAV capsid variants to deliver genesto cardiac cells.

Example 4

The cell tropism of recombinant AAV virions comprising the novel AAVvariants LANKIQRTDA+V708I (SEQ ID NO:43), LANKTTNKDA+V708I (SEQ IDNO:48), and AAV6/AAV5 chimera for skeletal myofibers was assessed invitro using skeletal myofibers generated from primary human myoblasts.

Recombinant AAV virions comprising either an AAV8 capsid, an AAV9capsid, the novel variant capsid LANKIQRTDA+V708I (SEQ ID NO:43), thenovel variant capsid LANKTTNKDA+V708I (SEQ ID NO:48), or the novelvariant capsid AAV6/AAV5 chimera and a genome comprising a greenfluorescent protein (EGFP) transgene operably linked to a CAG promoter(AAV8.CAG.EGFP, AAV9.CAG.EGFP, LANKIQRTDA+V708I (SEQ ID NO:43).CAG.EGFP,LANKTTNKDA+V708I (SEQ ID NO:48).CAG.EGFP, and AAV6/AAV5 chimera.CAG.GFP,respectively) were manufactured using standard methods. Skeletalmyofibers were generated from primary human skeletal myoblasts obtainedfrom a healthy 51 year old male (Cook Myosites). The myoblasts weredifferentiated for 30 days to form mature multinucleated skeletal musclefibers. The generated skeletal myofibers were evaluated for expressionof Myosin Heavy Chain (MHC) and Dystrophin to ensure that the majorityof the generated skeletal myofibers reached a mature state prior tovector characterization.

Relative to AAV8 and AAV9, the LANKIQRTDA+V708I (SEQ ID NO:43),LANKTTNKDA+V708I (SEQ ID NO:48), and AAV6/AAV5 chimera provided forsignificantly higher transduction efficiency of and transgene expressionin human skeletal myofiber cultures seven days post-infection asdetermined by immunofluorescence (FIG. 8A) and flow cytometry (FIG. 8B).Furthermore, relative to AAV8 and AAV9, LANKIQRTDA+V708I (SEQ ID NO:43)and LANKTTNKDA+V708I (SEQ ID NO:48) provided for faster onset of geneexpression in human skeletal myofiber cultures, as determined byimmunofluorescence (FIG. 8C). Relative to AAV8 and AAV9, the number ofinfectious units per administered viral genome were multiple foldmagnitude higher for LANKIQRTDA+V708I (SEQ ID NO:43), LANKTTNKDA+V708I(SEQ ID NO:48), and the AAV6/5 chimera (FIG. 10B). This studyillustrates the superior ability of NKIQRTD (SEQ ID NO:13)-, NKTTNKD(SEQ ID NO:14)-, and SEQ ID NO:62-comprising variants to deliver genesto skeletal myofibers.

Example 5

The cell tropism of recombinant AAV virions comprising the novel AAVvariants LANKIQRTDA+V708I (SEQ ID NO:43), LANKTTNKDA+V708I (SEQ IDNO:48), and AAV6/AAV5 chimera for skeletal muscle progenitor cells wasassessed in vitro using skeletal muscle progenitor cells generated fromfibroblast-derived human induced pluripotent stem cells (FB-iPSC) orhuman embryonic stem cells (ESC).

Recombinant AAV virions comprising either an AAV9 capsid, the novelvariant capsid LANKIQRTDA+V708I (SEQ ID NO:43), the novel variant capsidLANKTTNKDA+V708I (SEQ ID NO:48), or the novel variant capsid AAV6/AAV5chimera and a genome comprising a green fluorescent protein (EGFP)transgene operably linked to a CAG promoter (AAV8.CAG.EGFP,AAV9.CAG.EGFP, LANKIQRTDA+V708I (SEQ ID NO:43).CAG.EGFP,LANKTTNKDA+V708I (SEQ ID NO:48).CAG.EGFP, and AAV6/AAV5 chimera.CAG.GFP,respectively) were manufactured using standard methods. Skeletal muscleprogenitors were generated from a human embryonic stem cell line,ESI-017 (ESI-BIO) following the differentiation strategy described inShelton et al. Methods, 2016 with minor modifications. Afterapproximately 40 days of differentiation, lineage restriction toskeletal muscle progenitors was confirmed by expression of PAX7, andMyoD in the majority of cells prior to using the cultures for vectorcharacterization.

Relative to AAV9, the LANKIQRTDA+V708I (SEQ ID NO:43), LANKTTNKDA+V708I(SEQ ID NO:48), and AAV6/AAV5 chimera provided for significantly highertransduction efficiency of and transgene expression in human skeletalmuscle progenitor cultures six days post-infection as determined byimmunofluorescence (FIG. 9A) and flow cytometry (FIG. 9B). This studyillustrates the superior ability of NKIQRTD (SEQ ID NO:13)-, NKTTNKD(SEQ ID NO:14)-, and SEQ ID NO:62-comprising AAV capsid variants todeliver genes to skeletal muscle progenitors.

Example 6

Directed evolution was employed to discover novel adeno-associated virus(AAV) variants with superior gene delivery to cardiac and skeletalmuscle cells following intravenous (IV) administration, a route ofadministration with significant advantages over other methods of genedelivery to the human heart and skeletal muscle (Example 1). The celltropism following intramuscular administration of recombinant AAVvirions comprising the novel AAV variant comprising a V708I substitutionand the peptide LANKIQRTDA (SEQ ID NO:27) inserted between amino acids587 and 588 (LANKIQRTDA+V708I; SEQ ID NO:43) was assessed in vivo inmice as a representative example of the ability of rAAV virionscomprising NKIQRTD (SEQ ID NO:13)-containing AAV capsid variants totransduce muscle cells.

Recombinant AAV virions comprising the novel variant capsidLANKIQRTDA+V708I and a genome comprising a luciferase transgene operablylinked to a CAG promoter (LANKIQRTDA+V708I (SEQ IDNO:43).CAG.luciferase) were manufactured using standard methods. B6Albino (C57BL/6) mice were injected via tail vein intravenous injectionwith of 2×10² vg, and transduction was assessed in-life by luciferaseimaging and post-mortem by tissue luciferase activity. In life imagingof luciferase at day 14 (left) and day 28 (right) post-administrationdemonstrate that the novel AAV variant LANKIQRTDA+V708I (SEQ ID NO:43)capsid can transduce mouse cells in vivo (FIG. 11A). Luciferase activityin heart, diaphragm, and quadriceps 56 days post-administrationdemonstrate that the novel AAV variant LANKIQRTDA+V708I (SEQ ID NO:43)capsid can transduce mouse cardiac and skeletal muscle in vivo (FIG.11B).

This study illustrates gene delivery by the NKIQRTD (SEQ IDNO:13)-comprising variant following one of several clinically acceptableroutes of administration. Similar efficacy is achievable with othervariants comprising this peptide insertion motif. Likewise, similarefficacy is achievable with other variants disclosed herein that wereidentified using the same directed evolution approach.

Example 7

Directed evolution was employed to discover novel adeno-associated virus(AAV) variants with superior gene delivery to cardiac and skeletalmuscle cells following intravenous (IV) administration, a route ofadministration with significant advantages over other methods of genedelivery to the human heart and skeletal muscle (Example 1). The celltropism following intramuscular administration of recombinant AAVvirions comprising the novel AAV variant comprising a V708I substitutionand the peptide LANKIQRTDA (SEQ ID NO:27) inserted between amino acids587 and 588 (LANKIQRTDA+V708I; SEQ ID NO:43) was assessed in vivo innon-human primates (NHP) as a representative example of the ability ofrAAV variants comprising NKIQRTD (SEQ ID NO:13)-containing AAV capsidvariants to transduce muscle cells.

Recombinant AAV virions comprising the novel variant capsidLANKIQRTDA+V708I (SEQ ID NO:43) and a genome comprising a greenfluorescent protein (GFP) transgene operably linked to a CAG promoter(LANKIQRTDA+V708I (SEQ ID NO:43).CAG.GFP) were manufactured usingstandard methods. Cynomolgus macaques were injected via intramuscularinjection with three doses of vector into sites in the vastus lateralisof 1×10 vg and the transduction of skeletal muscle cells was assessedpost-mortem by immunofluorescence imaging. Representative images ofhaemotoxylin and eosin (H&E) and anti-GFP antibody staining ofcross-sections of the proximal biopsy site at 2×, 4×, and 20×magnification demonstrate that the novel AAV variant LANKIQRTDA+V708I(SEQ ID NO:43) capsid can transduce primate skeletal muscle cells invivo (FIG. 12A). Representative images of haemotoxylin and eosin (H&E)and anti-GFP antibody staining of longitudinal sections of the distalbiopsy site at 2×, 4×, and 20× magnification demonstrate that the novelAAV variant LANKIQRTDA+V708I (SEQ ID NO:43) capsid can transduce primateskeletal muscle cells in vivo (FIG. 12B).

This study illustrates gene delivery by the NKIQRTD (SEQ IDNO:13)-comprising variant following one of several clinically acceptableroutes of administration. Similar efficacy is achievable with othervariants comprising this peptide insertion motif. Likewise, similarefficacy is achievable with other variants disclosed herein that wereidentified using the same directed evolution approach.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions.

Moreover, all statements herein reciting principles, aspects, andembodiments of the invention as well as specific examples thereof, areintended to encompass both structural and functional equivalentsthereof. Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of the present invention isembodied by the appended claims.

What is claimed is:
 1. A variant adeno-associated virus (AAV) capsidprotein comprising the amino acid sequence set forth in SEQ ID NO:43 orcomprising the amino acid sequence set forth in SEQ ID NO:48.
 2. Thevariant AAV capsid protein of according to claim 1, wherein the AAVcapsid protein comprises the amino acid sequence set forth in SEQ IDNO:48.
 3. The variant AAV capsid protein of claim 1, wherein the AAVcapsid protein comprises or consists of the amino acid sequence setforth in SEQ ID NO:43.
 4. An isolated nucleic acid comprising anucleotide sequence that encodes a variant AAV capsid protein ofclaim
 1. 5. An infectious recombinant AAV (rAAV) virion comprising avariant AAV capsid protein of claim
 1. 6. The rAAV virion of claim 5further comprising a heterologous nucleic acid comprising a nucleotidesequence encoding a gene product.
 7. The rAAV virion of claim 6, whereinthe gene product is a protein, a small interfering RNA, an antisenseRNA, a microRNA, a short hairpin RNA or a small interfering RNA.
 8. TherAAV virion of claim 7, wherein the rAAV virion comprises a heterologousnucleic acid encoding a gene product selected from alpha galactosidase A(GLA), frataxin (FXN), dystrophin (DMD) or a functional fragmentthereof, acid alpha glucosidase (GAA), and glycogen phosphorylase,muscle (PYGM).
 9. The rAAV virion of claim 8, wherein the variant AAVcapsid protein comprises the amino acid sequence set forth in SEQ IDNO:48 and wherein the heterologous nucleic acid comprises a nucleotidesequence encoding an alpha galactosidase A (GLA) protein.
 10. The rAAVvirion of claim 9, wherein the variant AAV capsid protein comprises orconsists of the amino acid sequence set forth as SEQ ID NO:48 andwherein the nucleotide sequence encoding GLA is operably linked to a CAGpromoter.
 11. The rAAV virion of claim 10, wherein the variant AAVcapsid protein consists of the amino acid sequence set forth in SEQ IDNO:48.
 12. The rAAV virion of claim 8, wherein the variant AAV capsidprotein comprises the amino acid sequence set forth in SEQ ID NO:48 andwherein the heterologous nucleic acid comprises a nucleotide sequenceencoding a dystrophin protein or a functional fragment thereof.
 13. TherAAV virion of claim 8, wherein the variant AAV capsid protein comprisesthe amino acid sequence set forth in SEQ ID NO:48 and wherein theheterologous nucleic acid comprises a nucleotide sequence encoding afrataxin protein.
 14. The rAAV virion of claim 13, wherein the variantAAV capsid protein consists of the amino acid sequence set forth as SEQID NO:48 and wherein the nucleotide sequence encoding a frataxin proteinis operably linked to a CAG promoter.
 15. The rAAV virion of claim 8,wherein the variant AAV capsid protein comprises the amino acid sequenceset forth in SEQ ID NO:48 and wherein the heterologous nucleic acidcomprises a nucleotide sequence encoding a GAA protein.
 16. The rAAVvirion of claim 15, wherein the variant AAV capsid protein consists ofthe amino acid sequence set forth as SEQ ID NO:48 and wherein thenucleotide sequence encoding a GAA protein is operably linked to a CBApromoter.
 17. A pharmaceutical composition comprising an rAAV virion ofclaim 6 and a pharmaceutically acceptable excipient.
 18. A method ofdelivering a heterologous nucleic acid to a muscle cell comprisingcontacting the muscle cell with an rAAV virion of claim
 6. 19. Themethod of claim 18, wherein the muscle cell is a cardiac and/or skeletalmuscle cell.
 20. A method to treat Fabry disease in a subject in needthereof, comprising administering to the subject an effective amount ofan rAAV virion of claim 9 or a pharmaceutical composition comprising anrAAV virion of claim
 9. 21. A method to treat Duchenne's musculardystrophy in a subject in need thereof, comprising administering to thesubject an effective amount of an rAAV virion of claim 12 or apharmaceutical composition comprising an rAAV virion of claim
 12. 22. Amethod to treat Friedreich's ataxia in a subject in need thereof,comprising administering to the subject an effective amount of an rAAVvirion of claim 13 or a pharmaceutical composition comprising an rAAVvirion of claim
 13. 23. A method to treat Pompe disease in a subject inneed thereof, comprising administering to the subject an effectiveamount of an rAAV virion of claim 15 or a pharmaceutical compositioncomprising an rAAV virion of claim
 15. 24. The method of claim 20,wherein the rAAV virion or pharmaceutical composition is administered tothe subject by intravenous and/or intramuscular injection.
 25. Themethod of claim 21, wherein the rAAV virion or pharmaceuticalcomposition is administered to the subject by intravenous and/orintramuscular injection.
 26. The method of claim 22, wherein the rAAVvirion or pharmaceutical composition is administered to the subject byintravenous and/or intramuscular injection.
 27. The method of claim 23,wherein the rAAV virion or pharmaceutical composition is administered tothe subject by intravenous and/or intramuscular injection.
 28. Theisolated nucleic acid of claim 4, wherein the nucleic acid comprises anucleotide sequence that encodes a variant AAV capsid protein comprisingthe amino acid sequence set forth as SEQ ID NO:48.
 29. The infectiousrAAV virion of claim 5, wherein the rAAV virion comprises a variant AAVcapsid protein comprising the amino acid sequence set forth as SEQ IDNO:48.
 30. The pharmaceutical composition of claim 17, wherein the rAAVvirion comprises a variant AAV capsid protein comprising the amino acidsequence set forth as SEQ ID NO:48, and wherein the pharmaceuticalcomposition comprises from 1×10¹¹ to 1×10¹⁵ recombinant virions.
 31. Themethod of claim 24, wherein the nucleotide sequence encoding GLA isoperably linked to a CAG promoter.